Monitoring monomer-specific acyl-tRNA levels in cells with PARTI

Monitoring monomer-specific acyl-tRNA levels in cells with PARTI | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Monitoring monomer-specific acyl-tRNA levels in cells with PARTI View ORCID Profile Meghan A. Pressimone , View ORCID Profile Carly K. Schissel , View ORCID Profile Isabella H. Goss , View ORCID Profile Cameron V. Swenson , View ORCID Profile Alanna Schepartz doi: https://doi.org/10.1101/2025.01.14.633079 Meghan A. Pressimone 1 Department of Molecular and Cellular Biology, University of California , Berkeley, CA 94720, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Meghan A. Pressimone Carly K. Schissel 2 Department of Chemistry, University of California , Berkeley CA 94720, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Carly K. Schissel Isabella H. Goss 2 Department of Chemistry, University of California , Berkeley CA 94720, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Isabella H. Goss Cameron V. Swenson 2 Department of Chemistry, University of California , Berkeley CA 94720, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Cameron V. Swenson Alanna Schepartz 1 Department of Molecular and Cellular Biology, University of California , Berkeley, CA 94720, USA 2 Department of Chemistry, University of California , Berkeley CA 94720, USA 3 3 Institute for Quantitative Biosciences (QB3), University of California , Berkeley, CA 94720, USA 4 Chan Zuckerberg Biohub , San Francisco, CA 94158, USA 5 ARC Institute, Palo Alto , CA 94304, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alanna Schepartz For correspondence: schepartz{at}berkeley.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT We describe a new assay that reports directly on the acylation state of a user-chosen tRNA in cells. We call this assay 3-Prime Adenosine-Retaining Aminoacyl-tRNA Isolation (PARTI). It relies on high-resolution mass spectrometry identification of the acyl-adenosine species released upon RNase A cleavage of isolated cellular tRNA. Here we develop the PARTI workflow and apply it to understand three recent observations related to the cellular incorporation of non-α-amino acid monomers into protein: ( 1 ) the origins of the apparent selectivity of translation with respect to β 2 -hydroxy acid enantiomers; ( 2 ) the activity of PylRS variants for benzyl derivatives of malonic acid; and ( 3 ) the apparent inability of N -Me amino acids to function as ribosome substrates in living cells. Using the PARTI assay, we also provide direct evidence for the cellular production of 2’,3’-diacylated tRNA in certain cases. The ease and simplicity of the PARTI workflow should benefit ongoing efforts to study and improve the cellular incorporation of non-α-amino acid monomers into proteins. Download figure Open in new tab GRAPHICAL ABSTRACT INTRODUCTION There is widespread interest in the cellular biosynthesis of genetically encoded materials containing one or more non-α-amino acid monomers ( 1 ). Even monomers that differ from α-amino acids by a single atom, such as α-hydroxy acids, can generate proteins with unique properties. Examples include discrete amide to ester substitutions that probe ion channel function ( 2 ) or the contributions of backbone H-bonds to protein stability ( 3 ), and others that support intramolecular rearrangements to generate expanded or altered backbones ( 4 , 5 ). Over the past few years, a small number of non-α-amino acids, notably β 2 -hydroxy and β 3 -amino acids, have been introduced into proteins in cells using native machinery or orthogonal aminoacyl tRNA synthetase (aaRS)/tRNA pairs and stop codon suppression ( 6 – 8 ). Yet even with highly active or evolved aaRS enzymes, protein yields are low, unpredictable, or both ( 7 , 8 ). Although ribosomal translation is complex and multi-step, and myriad events could contribute to low or unpredictable protein yields ( 3 , 9 – 11 ), the level of tRNA aminoacylation is essential. A robust assay that reports directly on tRNA acylation levels in cells would streamline efforts to optimize the cellular incorporation of non-α-amino acid monomers into proteins. An assay that reports simultaneously on monomer identity would provide an even more accurate snapshot of the state of tRNA acylation and thus the specificity of the corresponding aaRS. The challenge in the development of an assay that reports simultaneously on cellular tRNA acylation and monomer identity is that expression of an aaRS/tRNA pair in the presence of an aaRS substrate can result in multiple different acyl-tRNA products ( Figure 1A ). These products include the expected 3’-monoacylated tRNA as well as those carrying a second acyl group on the 2’-hydroxyl groups as observed in vitro ( 7 , 12 , 13 ), and others that are misacylated with an incorrect or metabolized version of the aaRS substrate as observed in cells using protein translation as a proxy ( 3 , 9 , 14 ). Few methods directly capture the in vivo acylation level of a single tRNA of interest. Of those that do, most rely on differentiating acylated and unreacted tRNA populations using periodate oxidation and fail to distinguish between different potential acyl-tRNA products ( 15 – 18 ). Download figure Open in new tab Figure 1: 3-Prime Adenosine-Retaining aminoacyl-tRNA Isolation (PARTI) reports on monomer-specific tRNA acylation in cells. (A) Schematic showing three states of tRNA – monoacylated, misacylated, or diacylated – that can exist in E. coli expressing an aaRS/tRNA pair and supplemented with substrate. As per ISAP, the tRNA to be analyzed is selectively captured from total cellular RNA using a biotinylated complementary DNA oligonucleotide and sequestered using streptavidin-coated magnetic beads. (B) In the PARTI workflow, the sequestered tRNA population is treated with RNase A to release the 3’ aminoacyl adenosine (aa-Ade) which is then detected using LC-HRMS and quantified using a peptide standard, leucine-enkephalin (Leu-Enk). One recently reported method that provides a partial snapshot of the cellular tRNA acylation state, including the identity of the acylated monomer, is referred to as isoacceptor-specific affinity purification (ISAP) ( 19 ). Here, total RNA is first isolated from cells grown in the presence of an aaRS substrate and the tRNA of interest is extracted from total cellular RNA using a complementary biotinylated DNA oligonucleotide ( Figure 1B ). The population of acyl-tRNAs is subsequently hydrolyzed and the monomer detected by mass spectrometry ( 19 ). Although ISAP is highly sensitive, the post-capture hydrolysis step demands extensive washing to remove contaminating amino acids and leverages radiolabeling to detect low levels of misacylation, which is challenging for non-canonical monomers not available in radiolabeled form. Additionally, because it relies on hydrolysis of the acylated tRNA to release the subsequently detected monomer, it conflates tRNA populations that are mono- and diacylated. As a result, ISAP overestimates the acylation yield, and hence the activity, of any monomer that doubly acylates tRNA in cells, as has been observed for multiple backbone-modified monomers in vitro ( 7 , 9 , 12 ). Complicating matters further is the fact that tRNAs acylated with some non-α-amino acids resist hydrolysis ( 20 ), which underestimates acylation yield and activity. Here we describe a variation of ISAP that overcomes these limitations to provide a detailed snapshot of cellular tRNA acylation. Rather than relying on the detection of monomers after acyl-tRNA hydrolysis, here we detect the acylated 3’-adenosine using high resolution mass spectrometry (HRMS) after enzymatic cleavage of the terminal 3’-adenosine using RNase A ( Figure 1B ) ( 9 , 21 ). Detecting the 3’-adenosine by mass spectrometry unambiguously identifies the acylated species and whether the tRNA has been diacylated on both the 2’ and 3’ position. We refer to this new assay as PARTI, for 3- P rime A denosine- R etaining Aminoacyl-tRNA I solation ( Figure 1B ). Here we develop a robust PARTI workflow and apply it to address three outstanding questions related to the cellular synthesis of proteins containing one or more non-α-amino acid monomers: the enantioselectivity of pyrrolysyl tRNA synthetase (PylRS) in cells with respect to β 2 -hydroxy acid substrates ( 7 ), the cellular activity of PylRS variants with respect to benzyl malonate substrates ( 9 ), and the metabolic fate of N -methyl ( N- Me) amino acids in cells, which have been introduced into peptides and proteins, but only in in vitro translation mixtures and cell extracts ( 22 – 30 ). We anticipate that PARTI will aid the analysis of cellular tRNA acylation status and benefit ongoing efforts to expand the proteome to include diverse backbone-modified monomers. METHODS GENERAL METHODS Note: Procedures involving RNA were carried out using RNase-free materials and technique, which includes decontaminating the work area with RNase FREE (Apex Bio) or a similar product. Transcription and purification of in vitro tRNAs DNA templates used to transcribe either E. coli tRNA Phe ( Ec tRNA Phe ) or Methanomethylophilus alvus tRNA Pyl ( Ma tRNA Pyl ) were generated by annealing and extending the single-stranded DNA oligonucleotide pairs PheT-Fwd and PheT-Rev or PylT-Fwd and PylT-Rev (2 mM; Supplementary Information, Table S1 ) using OneTaq 2x Master Mix (NEB) according to the manufacturer protocol. Both Ec tRNA Phe and Ma tRNA Pyl were transcribed in vitro using the TranscriptAid Kit (NEB) according to the manufacturer protocol. Transcription reactions of a final volume of 200 µL were divided into eight 25 µL aliquots and incubated at 37 °C for 4 h. Then, the eight 25 µL reactions were pooled, 20 µL DNase I (NEB) was added, and the reaction was incubated at 37 °C for 1 h followed by purification using gel electrophoresis and ethanol precipitation as described ( 31 ). The tRNA was then analyzed for purity using intact tRNA LC-MS as described ( 32 ). A representative analysis of Ec tRNA Phe is shown in Supplementary Information, Figure S1. Cell Transformations Chemically competent cells in 100 µL aliquots were transformed by incubating with 100 ng plasmid DNA on ice for 30 min followed by a 90 sec heat-shock at 42 °C. The cells were allowed to recover on ice for 2 min, supplemented with 800 µL LB, grown at 37 °C for one hour with shaking, and applied to a LB/agar plate prepared with the appropriate antibiotic. Purification of aminoacyl-tRNA synthetases M. alvus PylRS ( Ma PylRS) was purified from BL21-Gold (DE3)pLysS chemically competent cells and analyzed as described ( 9 ) except that TALON resin (Takara Bio) was used rather than Ni-NTA agarose resin. The yield was 170 mg/L Ec PheRS was purified from BL21-Gold (DE3)pLysS chemically competent cells using the same protocol as for Ma PylRS ( 9 ) with the following changes. The plasmid used to express Ec PheRS was derived from pET-32a and contained the sequences of the α and β subunits of Ec PheRS; a His 6 -SUMO tag was encoded at the N-terminus of the αsubunit. Following purification with TALON resin, the protein concentration was determined using a NanoDrop ND-1000 device (Thermo Scientific). The His 6 -SUMO tag was removed by adding the purified protein and SUMO protease purified previously from BL21-Gold (DE3) cells transformed with a pET-32a plasmid containing the sequence for SUMO protease with a His 6 tag encoded at the N-terminus ( 33 ) in an 8:1 molar ratio to Snakeskin™ dialysis tubing (10K MWCO, Thermo) equilibrated with Storage Buffer (100 mM HEPES-K (pH 7.2), 100 mM NaCl, 10 mM MgCl2, 4 mM dithiothreitol (DTT), 20% (v/v) glycerol) and incubated for 16 h at 4 °C. The protein was then concentrated using an Amicon® Ultra Centrifugal Filter, 10 kDa MWCO and the buffer exchanged to 50 mM sodium phosphate (pH 7.4), 500 mM NaCl, 20 mM β-mercaptoethanol using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva). Ec PheRS was then isolated from the His 6 -SUMO tag by incubating the sample with TALON resin for 45 min at 4°C and collecting the flowthrough containing Ec PheRS. Ec PheRS was analyzed by LC-HRMS as described ( 9 ) ( Supplementary Information, Figure S1 ). Protein concentration was determined by Pierce™ Bradford Assay (Thermo Scientific). The yield was 88 mg/L. In vitro tRNA acylation and analysis Acylation of in vitro -transcribed and purified Ma tRNA Pyl and Ec tRNA Phe was performed as previously described ( 9 ) using 25 µM tRNA and 1.5-12.5 µM of the requisite aaRS for 2-4 h, as specified per each experiment. Purified Ma PylRS was used to acylate Ma tRNA Pyl with 10 mM of a given substrate: L-α-amino-Boc-lysine, (BocK) (Sigma Aldrich), L-α-methyl-amino-Boc-lysine ( N -Me-BocK), (S)-6-((tert-Butoxycarbonyl)amino)-2-hydroxyhexanoic acid (OH-BocK) (Accela Chembio), (R)-6-((tert-butoxycarbonyl)amino)-2-(hydroxymethyl)hexanoic acid ( (R)- β 2 -OH-BocK) (Enamine), or (S) -6-((tert-butoxycarbonyl)amino)-2-(hydroxymethyl)hexanoic acid ( (S)- β 2 -OH-BocK) (Enamine). Purified Ec PheRS was used to acylate Ec tRNA Phe with 10 mM L-phenylalanine (Phe) (Frontier Scientific). Samples were analyzed by intact tRNA LC-MS and RNase A/LC-HRMS as described ( 9 , 32 ). The major ion (m/z), corresponding peak area, and percent acylation for each replicate can be found in Supplementary Information, Table S3 . Statistical analysis Either a one-way ANOVA or two-tailed t test was performed for statistical comparisons. Exact parameters and results can be found in Supplementary Information, Table S2 . 2. PARTI Workflow Design of biotinylated DNA capture oligonucleotide o-Pyl The sequence of the capture oligonucleotide for tRNA Pyl , o-Pyl, was designed to match the length and predicted T m of the established capture oligonucleotide for tRNA Phe ( 19 ), referred to here as o-Phe, with slight changes related to potential differences in modified bases. It is not known whether Ma tRNA Pyl contains modified bases, but the homolog from M. barkeri is proposed to have a 4-thiouridine at position 8 and 1-methyl pseudouridine at position 50 ( 34 ). To avoid these positions, the o-Pyl sequence used for PARTI was complementary to residues 27 to 49 and encompasses the full anticodon loop and half the anticodon and T stems ( Supplementary Information, Table S1 ), with a total of 5 fewer bases (26 versus 31 bases) than o-Phe and a T m predicted to be 2 °C lower (62.8 °C versus 64.5 °C), as predicted using Benchling software. While the capture oligonucleotide used in ISAP was biotinylated on the 3’ end, o-Phe and o-Pyl as used in this work are biotinylated on the 5’ end and were purchased from IDT with this modification. Cell Growth . The lac-inducible plasmids pMEGA-PylRS-PylT or pMEGA-FRSA-PylT ( 9 ) were transformed into either DH5α, Top10 or C321.ΔA.exp (C321) ( 35 ) chemically competent E. coli cells. The cells were then plated on a LB/agar plate prepared with 0.1 mg/mL spectinomycin and incubated overnight at 37 °C. A single colony was chosen and used to inoculate 10 mL of LB containing spectinomycin (0.1 mg/mL) and the cells grown with shaking at 200 rpm for 16 h. This overnight culture was used to inoculate subsequent growths as described below. For isolation of tRNA Phe : 500 mL of LB containing 0.1 mg/mL spectinomycin was inoculated with 5 mL of the overnight culture and grown at 37 °C with shaking at 200 rpm until the OD 600 reached 0.4–0.6. Expression of tRNA Pyl and PylRS was not induced. Cultures were pelleted by centrifugation using a Beckman Coulter Allegra®X-14R Benchtop Centrifuge using a Beckman SX4750 Swinging Bucket Rotor, frozen at -80 °C and stored for up to a month before RNA extraction as described below. For isolation of tRNA Pyl : 50 mL (unless otherwise noted) of LB containing 0.1 mg/mL spectinomycin was inoculated with 5 mL of the overnight culture, supplemented with aaRS substrate (final concentration 0.1-1 mM), and grown at 37 °C with shaking at 200 rpm. Cultures were grown until the OD 600 reached 0.4–0.6 then were supplemented with IPTG to a final concentration of 1 mM to induce the expression of aaRS and tRNA Pyl genes. The induced cultures were grown for 3 h at 37 °C with shaking at 200 rpm, then pelleted by centrifugation using a Beckman Coulter Allegra®X-14R Benchtop Centrifuge using a Beckman SX4750 Swinging Bucket Rotor and frozen at -80 °C. Pellets were stored for up to a month before RNA extraction as described below. Total RNA Extraction Samples were maintained on ice during all extraction steps. TRIzol™ Reagent (Thermo Scientific) was used and the extraction executed according to the manufacturer’s protocol. 3 mL Trizol was added to pellets originating from 500 mL uninduced log phase cells grown for tRNA Phe pulldown and 1 mL Trizol was added to pellets originating from 50 mL cultures expressing tRNA Pyl . Samples were mixed by vortexing and incubated on ice for 15 min to lyse the cells. Then 0.2 mL chloroform was added for every 1 mL Trizol, and a liquid-liquid extraction was performed. The aqueous layer containing total RNA was transferred to new 1.7 mL Eppendorf tubes and 0.5 mL isopropanol was added for every 1 mL Trizol. Samples were mixed by inverting the tubes then incubated on ice for 15 min to precipitate the RNA. Samples were centrifuged in a Eppendorf™ 5425 Centrifuge for 30 min at 21,000 relative centrifugal force (rcf) and 4 °C to pellet the RNA. The supernatant was discarded and the pellet was washed with 500 µL 70% ethanol and centrifuged for 10 min at 21,000 rcf and 4 °C. The supernatant was discarded and the RNA pellet dried at RT for 10 min. The pellet was either frozen at -80 °C or resuspended immediately. When ready to use, dried pellets were resuspended in 200 µL RNase free water (Milli Q). RNA concentration was determined by measuring the absorbance at 260 nm using a NanoDrop ND-1000 device (Thermo Scientific) and samples were kept on ice before tRNA isolation using o-Phe or o-Pyl. tRNA isolation using o-Phe or o-Pyl The following steps are adapted from Mohler et al. ( 19 ), save for the RNA quantity used. In a new 1.7 mL Eppendorf tube, 250 µg resuspended total RNA was mixed with 5 µL 100 µM biotinylated DNA oligomer (IDT) (500 pmol, final concentration 1 µM), 250 µL 4x saline-sodium citrate (SSC) (Invitrogen) pH 4.8 (final concentration 2X), and MilliQ RNase free water up to a final volume of 500 µL. The solution was incubated for 60 min at 50 °C on a Thermolyne Dri-Bath to generate the tRNA-DNA hybrid and then cooled to RT. While the tRNA-DNA hybrid solution was cooling to RT, 0.5 mg Streptavidin MagneSphere® Paramagnetic Particles (SA-PMPs) (Promega) (500 µL of a 1 mg/mL resuspension) were added to a fresh 1.7 mL tube. The maximum capacity of 0.5 mg of streptavidin-coated magnetic beads is estimated to be 625 pmol according to the Product Technical Bulletin. The tube was secured in a magnetic rack (MagRack 6, Cytiva) and the supernatant was removed carefully to not include any streptavidin-coated magnetic beads. The beads were washed 3 times with 300 µL 2x SSC pH 4.8 and the supernatant discarded before the tRNA-DNA hybrid solution was added to the beads and rotated for 10 min. The supernatant was then discarded, and the beads were washed 6 times with 300 µL 2x SSC pH 4.8. Finally, the beads were resuspended in 10 µL 2x SSC pH 4.8. RNase A cleavage and addition of MS standard Note: When using RNase A, special care must be taken to avoid contamination of surfaces, pipettes, etc. Surfaces should be decontaminated with RNase FREE (Apex Bio) or a similar product. 11 µL 1.51 U/µL RNase A (VWR) dissolved in 200 mM NaOAc was added to the resuspended beads prepared as described above, and the mixture was incubated for 10 min at RT ( 21 ). 2.2 µL 50% TCA was added and the sample was incubated for an additional 10 min to halt the RNase A cleavage reaction ( 21 ). The samples were centrifuged for 10 min at 21,000 rcf and RT and a 20 µL aliquot of the supernatant was transferred to a new 1.7 mL tube and supplemented with 2 µL of a 40 ng/µL solution of leucine-enkephalin (Leu-Enk) (Waters). This solution diluted 1:10 with Mobile Phase A (0.1% formic acid in water) into a SureSTART™ 0.3 mL Glass Screw Top Microvial (Thermo Scientific) before LC-HRMS analysis as described below. Because the streptavidin-coated magnetic beads can retain an inconsistent volume of 2x SSC from wash steps during tRNA isolation, the remaining volume of supernatant beyond the 20 µL used for LC-HRMS was recorded for quantitatively determining A norm . The equation for determining A norm is provided below. LC-HRMS and Analysis 1 µL of the sample prepared as described above was analyzed by LC-HRMS as described ( 9 ), with the following modifications. Chromatography was performed at a flow rate of 0.5 mL/min using mixtures of Mobile Phase B (100% acetonitrile) and Mobile Phase A (0.1% formic acid in water). For each injection at t = 0, the eluent was initially held at 4% Mobile Phase B for 1.89 min. The amount of Mobile Phase B was then increased linearly from 4 to 40% over 3.11 min, then from 40 to 100% Mobile Phase B over 2 min, from 100 to 4% Mobile Phase B over 2 min, and finally held at 4% Mobile Phase B for 0.5 min to complete the chromatographic run. Mass spectrometry data was collected as described ( 9 ) between 1.4 and 7 min, with m/z ranging 100-3000 recorded. A norm values were determined as follows: First, the presence of free Ade or a given mono- or diacyl adenosine species was confirmed by generating an extracted ion chromatogram (EIC) of the expected exact [M+H] to five decimal places, determined using ChemDraw software, over a symmetric range of 100 ppm. Free Ade yielded one peak. aa-Ade appeared as two EIC peaks corresponding to the 2′- and 3′-acyl-adenosine products for chiral monomers or as more peaks for prochiral monomers. Di-aa-Ade, when observed, appeared as a single peak in the respective EIC. Chromatographic peaks containing the exact mass (error < 5 ppm) as well as the expected mass envelope were counted. Then, for quantification, new EICs were generated by extracting over the m/z range of the monoisotopic peak present in the sample. This process was repeated for Leu-Enk in the sample. The integrated areas of each analyte peak were recorded and the following equation was used to generate the corresponding A norm value: 3. METHODS IN SUPPORT OF INDIVIDUAL FIGURES Methods in Support of Figure 2 In vitro acylation reactions were performed using 10 mM Phe, 25 µM tRNA Phe and 1.5 µM purified E. coli PheRS in a final volume of 25 µL for 2.5 h at 37 °C and was otherwise the same as described in the methods for in vitro acylation. From a 15 µL aliquot of the reaction, Phe-tRNA Phe was isolated by phenol chloroform extraction and ethanol precipitation then resuspended in water. Intact-tRNA LC-MS was carried out as described previously with 10 pmol of sample ( 32 ). The remaining 250 pmol of the tRNA Phe acylation in 10 µL was treated with RNase A as described. LC-HRMS was carried out as described except for the length of run time and MS collection window. Chromatography was carried out with mobile phase B at 4% for 1.89 min followed by a linear gradient from 4-40% over 1.75 min. Then, mobile phase B underwent a gradient from 40-100% over 0.56 min and a subsequent gradient from 100-4% over 0.98 min. Mobile phase B was held at 4% for an additional 1.12 min. Mass spectrometry data were collected for the entire duration of the LC-HRMS run. In vivo Phe-tRNA Phe was purified first by growing E. coli DH5α cells transformed with a pET32a plasmid containing a carbenicillin resistance gene and a T7-promoted Ma PylRS gene; the latter was not expressed because DH5α cells do not express T7 RNA polymerase ( 36 ). The PARTI protocol was carried out as described but with 95 µg starting total RNA. LC-HRMS analysis was performed in the same manner as for the i n vitro Phe-tRNA Phe sample described in this section. Methods in Support of Figure 3 PARTI was carried out as described for purification of tRNA Phe from E. coli DH5α cells transformed with, but not expressing, pMEGA-PylRS-PylT. Methods in Support of Figure 4 An in vitro acylation was carried out as described with 25 µM in vitro -transcribed and purified tRNA Pyl , 10 µM purified M. alvus PylRS, and 10 mM BocK in 50 µL for 2 hours at 37°C. BocK-tRNA Pyl was then purified using the RNA Clean & Concentrate Kit (Zymo) and the concentration was determined using a NanoDrop ND-1000 device (Thermo Scientific). Then, PARTI was carried out using o-Pyl as described with 500 pmol total tRNA Pyl . In parallel, 10 pmol of total tRNA Pyl was analyzed by intact RNA LC-MS to determine the acylation yield. Methods in Support of Figure 5 PARTI was carried out as described for purification of tRNA Pyl from E. coli C321 cells transformed with pMEGAPylRS/PylT and incubated with no substrate or 0.1 mM either BocK, OH-BocK, ( S) -β 2 -OH-BocK, or ( R) -β 2 -OH-BocK. Methods in Support of Figure 6 PARTI was carried out as described for purification of tRNA Pyl with the following modifications. 30 mL of LB containing 0.1 mg/mL spectinomycin was inoculated with 3 mL of an overnight culture of E. coli C321 cells transformed with pMEGA-FRSA/PylT and supplemented with either no substrate or 1 mM of 3-Bromo-L-phenylalanine ( m- Br-Phe) (CombiBlocks), or of previously synthesized ( 9 ) 2-(3-trifluoromethyl)malonic acid ( m -CF 3 -bma), (S)-2-amino-3-(3-bromophenyl)propanoic acid ( m -Br-bma), or 2-(3-methylbenzyl)malonic acid. Methods in Support of Figure 7 PARTI was carried out as described for purification of tRNA Pyl with E. coli Top10 cells transformed with pMega-PylRS/PylT were grown with either no substrate, 1 mM BocK, or 1 mM N-Me BocK. Expression, Purification, and LC-HRMS analysis of sfGFP-200TAG The expression, purification, and LC-HRMS analysis of sfGFP-200TAG was performed as described ( 9 ) with the following changes. Chemically competent E. coli Top10 cells were transformed with sfGFP-200TAG and pMega- Ma PylRS. TB media was used for outgrowth during sfGFP-200TAG expression and cultures were supplemented with either 1 mM BocK or 1 mM N -Me BocK. Yields were 36 mg/L when expression was supplemented with 1 mM BocK and 8 mg/L when expression was supplemented with 1 mM N -Me BocK. Preparation of N -Me BocK N -Me BocK was prepared from Fmoc- N -Me-Lys(Boc)-OH (Sigma Aldrich) using 20% piperidine in DCM as described ( 37 ). In brief, the reaction was stirred for 1.5 h and was determined to be complete by LC-MS. N -Me BocK was extracted three times with water and washed with DCM. The aqueous layer was flash frozen with dry ice/acetone and lyophilized for 5 days resulting in a white powder in quantitative yield. Small Molecule MS Analysis Mixtures of N -Me BocK and BocK at 0.1 mg/mL (999:1, 99:1, and 90:10 I-Me BocK:BocK) were prepared and analyzed using a Waters SQD2 mass spectrometer fitted with a reverse-phase C18 column, a 200-800 nm UV/vis detector, a 300-1000 nm fluorescence detector. 1 µL of each mixture was injected. Chromatography was performed at a flow rate of 0.5 mL/min using mixtures of Mobile Phase B (0.1% formic acid in acetonitrile) and Mobile Phase A (0.1% formic acid in water). For each injection at t = 0, the eluent was initially set to 10% Mobile Phase B and was then increased linearly from 10% to 95% over 3.5 min, then decreased from 95% to 10% Mobile Phase B for 0.1 min, and finally held at 10% Mobile Phase B for 1.5 min to complete the chromatographic run. Mass spectrometry data with M/z ranging from 150-800 Da was collected in positive mode and in centroid form with the following parameters: cone voltage = 30 V, probe temperature = 20 °C, scan time = 0.25 s. Plate reader analysis of sfGFP expression The protocol was carried out as described ( 9 ) with the following changes. Following transformation into E. coli Top10 or C321 cells and growth of starter cultures as described, 500 µL each culture was used to inoculate 25 mL TB supplemented with 0.1 mg/mL carbenicillin and 0.1 mg/mL spectinomycin in 250 mL baffled Erlenmeyer flasks. Cultures were grown to OD 0.6-0.8 before induction with IPTG (final concentration 1 mM). 180 µL induced culture was added to each well with 20 µL LB for no substrate control, or 20 µL substrate for final concentrations of either 1 mM N -Me BocK, 5 mM N -Me BocK, or 0-1 mM BocK. Substrates were dissolved in water and NaOH in equal molar amount to the monomer itself to ensure solubilization. RESULTS RNase A cleavage and LC-HRMS provide an accurate measure of tRNA acylation To develop a robust PARTI workflow, we first set out to confirm that RNase A treatment of in vitro -acylated tRNA followed by LC-HRMS analysis provides an accurate measure of tRNA acylation. We began with a well-characterized reaction, that of E. coli PheRS with E. coli tRNA Phe and phenylalanine, to generate Phe-tRNA Phe . Purified, in vitro -transcribed tRNA Phe was acylated using purified PheRS and the products analyzed using both intact tRNA LC-MS ( 32 ) ( Figure 2A ) and RNase A digestion followed by LC-HRMS ( 21 ) ( Figure 2B ). Intact tRNA LC-MS of the acylation reaction mixture revealed two RNA products; one whose deconvoluted mass corresponds to unreacted tRNA Phe and the other to tRNA Phe monoacylated with Phe (Phe-tRNA Phe ). Integration of the two peaks indicated an acylation yield of 61% ( Figure 2A ) ( 32 ). Download figure Open in new tab Figure 2: Validating the PARTI workflow in vitro and in cells using E. coli PheRS and tRNA Phe (A) Intact tRNA LC-MS analysis of the products resulting from in vitro acylation of tRNA Phe with Phe and PheRS. The total ion chromatogram (TIC) of the reaction mixture is shown along with the deconvoluted mass spectrum of each peak. The gray peak in the TIC corresponds to the deconvoluted spectrum in gray, with the expected and observed mass of unreacted tRNA Phe shown. The teal peak in the TIC corresponds to the deconvoluted spectrum in teal, with the expected and observed mass of Phe-tRNA Phe . The yield of Phe-tRNA Phe was determined by the ratio of the integrated areas of the major ion for Phe-tRNA Phe and unreacted tRNA Phe as previously described ( 32 ). (B) Extracted ion chromatograms (EICs) from LC-HRMS analysis of the products resulting from in vitro acylation of tRNA Phe with Phe and PheRS following RNase A treatment. The trace in black is the EIC corresponding to the M+H for adenosine (Ade), whereas the trace in teal corresponds to the M+H for Phe-Ade, whose structure as a mixture of 2’ and 3’ isomers is shown. No diacyl-tRNA was detected. The EIC peak area for Ade and the summed areas of the two peaks due to Phe-Ade were used to calculate an acylation yield of 74%. (C) The secondary structure of E. coli tRNA Phe bound to o-Phe, the biotinylated DNA capture oligonucleotide used to extract total tRNA Phe from isolated total RNA. (D) Schematic of purification of tRNA Phe from cells using o-Phe followed by RNase A cleavage and LC-HRMS analysis of the reaction products. (E) Overlaid traces showing the extracted ions for adenosine (Ade) in gray and phenylalanine-adenosine (Phe-Ade) in teal generated by applying the PARTI workflow to tRNA Phe isolated from E. coli. The acylation yield is calculated to be 56% using the strategy described in (B) . RNase A digestion of the acylation reaction followed by LC-HRMS also revealed two products: one whose mass corresponds to adenosine (Ade), the product expected from digestion of unreacted tRNA Phe , and the other (Phe-Ade) whose mass corresponds to the product expected from digestion of Phe-tRNA Phe ( Figure 2B ). The ion chromatogram extracted for the mass of Phe-Ade (EIC) contains two peaks at ∼3 min which we assign to the anticipated mixture of 2’ and 3’-acylated products ( 9 ). The ion chromatogram extracted for the mass of Ade contains a single peak near 1 min and two small peaks at the same retention time as Phe-Ade which result from fragmentation of Phe-Ade during MS. Integrating the Phe-Ade and Ade EIC peaks indicates an acylation yield of 74%, which agrees reasonably well with the 61% yield determined by intact tRNA LC-MS. No diacyl-tRNA Phe was observed under these conditions. Next, we evaluated the extent of tRNA Phe acylation in E. coli by sequestering the complete cellular population of acylated and unreacted cellular tRNA Phe using the validated biotin-tagged capture oligonucleotide o-Phe ( Figure 2C and Supplementary Information, Table S1 ) ( 19 ). Although E. coli contains two genes encoding tRNA Phe , the resulting tRNA sequences are identical ( 38 ). The sequestered total tRNA Phe was treated with RNase A to liberate the 3’-terminal adenosine and the reaction products were analyzed by LC-HRMS ( Figure 2D ). When extracted for the mass of Phe-Ade, the resulting ion chromatogram consists of two major peaks whose retention times and mass spectra match those detected in vitro and correspond to the 2’ and 3’-isomers of Phe-Ade ( Figure 2E and Supplementary Information, Figure S2 ). When extracted for the mass of Ade, the ion chromatogram consists of a single major peak whose retention time also matches that of the in vitro sample ( Figure 2E and Supplementary Information, Figure S2 ). Diacylation was not observed, and integration of the Phe-Ade and Ade EIC peaks indicate an in vivo acylation yield of 56% ( 39 ). No Phe-Ade was detected when the total RNA was extracted using o-Pyl, a biotin-tagged DNA oligonucleotide complementing tRNA Pyl rather than tRNA Phe , or when RNase A was withheld ( Supplementary Information, Figure S3, Table S1 ). These control experiments verify that RNase A can be used to assess the tRNA acylation level of material isolated from cells using biotin-tagged capture oligonucleotides ( 19 ). Controls and internal standard facilitates comparisons across PARTI samples To compare acylated tRNA levels across multiple samples, we modified the PARTI analysis to include an internal standard, the peptide leucine-enkephalin (Leu-Enk) ( 40 ). To establish the improved precision provided by the internal Leu-Enk standard, we again isolated total RNA from E. coli and captured the tRNA Phe population using o-Phe and streptavidin-coated magnetic beads as described. In this case, a fixed amount of Leu-Enk was added to each sample after RNase A cleavage and before LC-HRMS analysis. EIC traces for the expected masses of Phe-Ade and Leu-Enk show two Phe-Ade peaks, as expected, and one Leu-Enk peak with a retention time of 4.5 min ( Figure 3A ). The combined area of the Phe-Ade peaks was normalized to that of Leu-Enk to generate a unitless normalized value, A norm . We note that for established pulldown conditions using 250 µg total RNA, A norm values were consistent between five biological replicates over two days, whereas the raw Phe-Ade peak areas varied by data collection day, indicating the benefit of the Leu-Enk internal standard ( Figure 3B , C ). Download figure Open in new tab Figure 3: Controls and an internal standard facilitate comparisons across PARTI samples. ( A) Shown are overlaid extracted ion chromatograms (EICs) of Leu-Enk (gray, calc [M+H]: 556.2766 Da) and Phe-Ade (green, calc [M+H]: 415.1724 Da). (B) Shown is a bar graph comparing raw Phe-Ade peak area sums collected by LC-HRMS over different days. Each point shown corresponds to a biological replicate and error bars represent one standard deviation from the average. Data acquired on Day 1 is shown in dark green (mean = 2.36 x 10 6 ; SD = 3.68 x 10 6 ; n = 3), whereas data acquired on Day 2 is shown in turquoise (mean = 1.10 x 10 6 ; SD = 2.11 x 10 5 ; n = 2). Statistical analysis bars represent the results of a two-tailed t-test. P > 0.05 = ns; p ≤ 0.05 = *; p ≤ 0.01 = **. (C) Shown is a bar graph comparing Phe-Ade peak areas normalized to Leu-Enk in each sample collected by LC-HRMS over different days. Samples shown are the same as in (B). Each point shown corresponds to a biological replicate and error bars represent one standard deviation from the average. Data from Day 1 is shown in dark green (mean = 0.18; SD = 0.00; n = 3), whereas data from Day 2 is shown in turquoise (mean = 0.20; SD = 0.00; n = 2). Statistical analysis bars represent the results of a two-tailed t-test. p > 0.05 = ns; p ≤ 0.05 = *; p ≤ 0.01 = **. (D) Shown is a bar graph displaying A norm values for Phe-Ade detected using PARTI. Each point shown corresponds to a biological replicate and error bars represent one standard deviation from the average. Shown in green are A norm values for Phe-Ade determined under standard conditions: 250 µg total RNA per sample, 5 mg streptavidin-coated magnetic beads, and 500 pmol o-Phe, as described in Methods (mean = 0.19; SD = 0.01; n = 5). Shown in red are A norm values for Phe-Ade determined when total RNA per sample = 125 µg (mean = 0.15; SD = 0.02; n = 3). Shown in purple are A norm values when total RNA per sample = 500 µg (mean = 0.33; SD = 0.02; n = 3). Shown in blue are A norm values determined using 500 pmol o-Pyl (mean = 0; SD = 0; n = 3). Shown in orange is the A norm value determined using 1 nmol o-Phe (mean = 0.29; SD = 0.14; n = 3). Shown in black is is the A norm value determined when 1 mg (2x) streptavidin beads were used (mean = 0.07; SD = 0.02; n = 3). Statistical analysis bars represent the results of a two-tailed t-test. p > 0.05 = ns; p ≤ 0.05 = *; p ≤ 0.01 = **; p ≤ 0.001 = ***. In addition to including an internal standard, we also performed experiments to evaluate the dependence of A norm on multiple experimental parameters, including the concentrations of total RNA and the biotinylated DNA capture oligonucleotide as well as the mass of streptavidin-coated magnetic beads ( Figure 3D ). Compared to A norm values obtained from the established conditions shown in green, doubling the streptavidin bead mass reduced the A norm value by 63%, perhaps due to inefficient RNase A cleavage. Doubling the o-Phe concentration had no effect, providing evidence for near quantitative tRNA extraction ( Figure 3D ). We also observed that the A norm value was 74% higher when the total RNA was doubled and 21% lower when the total RNA was halved relative to the standard value of 250 µg ( Figure 3D ). This finding implies that a total RNA mass of 250 µg does not saturate the volume of beads used. Capture using an oligomer complementary to tRNA Pyl instead of o-Phe, or capture with o-Phe but without RNase A, yielded no Phe-Ade signal ( Figure 3D ). Validating o-Pyl as a capture oligonucleotide to sequester tRNA Pyl from cells With a validated PARTI workflow in place, we sought to apply it to evaluate the reactivity of an orthogonal tRNA/aaRS pair in cells supplemented with non-canonical α- and non-α-amino acids. We chose PylRS from M. alvus , which acylates M. alvus tRNA Pyl with α-hydroxy acids and β 2 -hydroxy acids carrying appropriate side chains ( 7 , 9 , 41 ). To capture the complete tRNA Pyl population, we designed a new biotinylated DNA capture oligonucleotide (o-Pyl) that is complementary to nucleotides 27 through 48 of tRNA Pyl ( Figure 4A and Supplementary Information, Table S1 ). To test the utility of o-Pyl, tRNA Pyl was transcribed in vitro , purified, and acylated with L-α-boc-lysine (BocK) using purified M. alvus PylRS ( Figure 4A , B ). The tRNA Pyl population was captured with o-Pyl, sequestered using streptavidin-coated magnetic beads, and treated with RNase A. The final sample generated using the PARTI protocol was doped with Leu-Enk and analyzed using LC-HRMS. Under these conditions, the yield of BocK-tRNA Pyl is 37% in line with previous reports ( 7 ) ( Supplementary Information, Figure S4 ). As expected, the EICs revealed the presence of Leu-Enk and BocK-Ade bound to the 2’ or 3’ end of adenosine (BocK-Ade) ( Figure 4B ). Only unreacted and monoacyl tRNA, but no diacyl tRNA, was detected, as observed previously ( 7 ) ( Supplementary Information, Figure S4 ). Download figure Open in new tab Figure 4: Validating PARTI in vitro and in cells using M. alvus PylRS/tRNA Pyl and BocK. (A) The secondary structure of M. alvus tRNA Pyl bound to o-Pyl and the structure of BocK. (B) In vitro acylation of tRNA Pyl with PylRS and BocK followed by PARTI analysis produces the extracted ion chromatograms (EICs) shown at right. The trace in indigo represents the EIC for the mass of BocK-Ade; the trace in gray represents the EIC for the mass of Leu-Enk. (C) Acylation of tRNA Pyl in cells followed by PARTI analysis produces the extracted ion chromatograms (EICs) shown at right. The four traces represent samples from E. coli DH5α cells expressing the M. alvus PylRS/tRNA Pyl pair and grown with or without BocK and extracted with either o-Phe or o-Pyl. BocK-Ade (calc [M+H]: 496.2514 Da) is detected only in the presence of BocK and when the total RNA is extracted with o-Pyl. Each trace is normalized to Leu-Enk (dashed line, calc [M+H]: 556.2766 Da) in each sample. Next, we sought to establish that A norm values were dependent on the amount of sequestered tRNA Pyl subjected to the PARTI workflow. To do so, we chose a monomer that results in detectable levels of both mono- and diacyl-tRNA Pyl in vitro in the presence of PylRS – L-α-hydroxy-Boc-lysine (OH-BocK) ( 7 , 9 ). In vitro -transcribed and purified tRNA Pyl was acylated with OH-BocK in the presence of PylRS to yield a mixture of unreacted, monoacylated, and diacylated tRNA Pyl ( Supplementary Information, Figure S5A-D ). Varying amounts of the mixed tRNA Pyl population were sequestered with o-Pyl, treated with RNase A, and analyzed by LC-HRMS ( Supplementary Information, Figure S5E ). We found that A norm values for detection of either acylated adenosine species trend linearly with the amount of starting tRNA Pyl ( Supplementary Information, Figure S5F ). As a final control, we grew E. coli expressing the M. alvus PylRS/tRNA Pyl pair in the presence or absence of the PylRS substrate BocK. We extracted total RNA, sequestered the tRNA Pyl population with o-Pyl and streptavidin-coated magnetic beads, treated the beads with RNase A, and analyzed the products using LC-HRMS ( Figure 4C ). Total RNA was also extracted with o-Phe. When BocK and o-Pyl were both present, BocK-Ade was detected alongside Leu-Enk in the EIC at the same retention time as in the in vitro control ( Figure 4 B, C ). In contrast, when BocK was present but o-Phe was used, or when BocK was absent and either o-Pyl or o-Phe were used, the BocK-Ade signal was absent ( Figure 4C ). PARTI reveals a difference in cellular acylation of tRNA Pyl with (R) - and (S) -β 2 -hydroxy acids Next, we applied PARTI to evaluate the acylation state of tRNA Pyl in cells expressing PylRS and supplemented with either (R)- or (S) -β 2 -hydroxy-boc-lysine (β 2 -OH-BocK) ( Figure 5A ). Download figure Open in new tab Figure 5: (R)- and (S)- β 2 -hydroxy acids are not equivalent substrates for PylRS in cells. (A) Structures of OH-BocK, ( R) -β 2 -OH-BocK, and ( S) -β 2 -OH-BocK. ( B) Shown is a bar graph displaying the relative A norm values when 0.1 mM of each monomer in (A) is added to E. coli C321 cells expressing the Ma PylRS/tRNA Pyl and PylRS and subjected to the PARTI workflow. Points shown correspond to biological replicates. Error bars represent one standard deviation from the average. Data representing the detection of OH-BocK-Ade is shown in pink (mean = 2.76; SD = 0.56; n = 3), detection of ( R) -β 2 -OH-BocK-Ade is shown in purple (mean = 2.18; SD = 0.32; n = 3) and detection of ( S) -β 2 -OH-BocK-Ade is shown in green (mean = 0.20; SD = 0.11; and n = 3). No β 2 -OH-BocK-Ade was observed when no substrate was added, as shown in gray (mean = 0.0; SD = 0.0; n = 3). Statistical analysis bars represent the results of a one-way ANOVA. p > 0.05 = ns; p ≤ 0.05 = *; p ≤ 0.01 = **; p ≤ 0.001 = ***. (C) Overlaid extracted ion chromatograms (EICs) of monoacyl-3’ Ade species detected by LC-HRMS following PARTI with E. coli C321 cells expressing the Ma PylRS/tRNA Pyl pair and grown with no substrate or 0.1 mM OH-BocK, ( R) -β 2 -OH-BocK, or ( S) -β 2 -OH-BocK. Traces are normalized to the Leu-Enk EIC in each sample, shown in dashed gray. (D) Overlaid extracted ion chromatograms (EICs) of diacylated 3’ Ade species detected by LC-MS following PARTI with E. coli C321 cells expressing tRNA Pyl and PylRS grown with no substrate or 0.1 mM OH-BocK, ( R) -β 2 -OH-BocK or ( S) -β 2 -OH-BocK. Traces are normalized to the Leu-Enk EIC in each sample, shown in dashed gray. Previous work has shown that both β 2 -OH-BocK stereoisomers are substrates for the M. alvus PylRS/tRNA Pyl pair in vitro , but only one stereoisomer– (R)- β 2 -OH-BocK–is incorporated into proteins in cells ( 7 ). In addition, although both β 2 -OH-BocK enantiomers are processed by PylRS in vitro as well as OH-BocK, under comparable conditions the yield of protein containing (R)- β 2 -OH-BocK was approximately ten-fold lower than the yield of protein containing OH-BocK ( 7 ). We used PARTI to determine whether the level of tRNA Pyl acylation by these monomers in cells could account for either or both of these observations. We grew E. coli C321 cells expressing the Ma PylRS/tRNA Pyl pair in the presence of 0.1 mM (R)- or (S) -β 2 -OH-BocK or OH-BocK ( Figure 5A ), extracted total RNA, and isolated the tRNA Pyl population using o-Pyl and streptavidin-coated magnetic beads. The beads were treated with RNase A and the eluted products characterized using LC-HRMS ( Figure 5B , C and Supplementary Information, Figure S6, S7 ). Under these expression conditions, which are identical to those used for cellular experiments reported previously ( 7 ), the extent of tRNA Pyl acylation depends on both β 2 -OH-BocK stereochemistry and backbone identity (α- or β) ( Supplementary Information, Figure S8 ). When the growths were supplemented with ( R) -β 2 -OH-BocK, the A norm value resulting from monoacylation of tRNA Pyl by ( R) -β 2 -OH-BocK-Ade was comparable to that of OH-BocK-Ade ( Figure 5B ), in line with the reported yields of these two acylated tRNAs in vitro ( 7 ). However, when the growths were supplemented with ( S) -β 2 -OH-BocK, the A norm value for ( S) -β 2 -OH-BocK-Ade was approximately 10–fold lower than that for either OH-BocK-Ade or (R) -β 2 -OH-BocK-Ade ( Figure 5B ) . These observations indicate that in cells, only a relatively small fraction of the available tRNA Pyl is acylated with ( S) -β 2 -OH-BocK. The first implication of this finding is that the previously reported selectivity for incorporation of (R) -β 2 -OH-BocK in cells is due at least in part to differences in tRNA Pyl acylation. Either (S) -β 2 -OH-BocK is a poor PylRS substrate in cells, or it is metabolized into an unknown product that is no longer a PylRS substrate. The second implication is that the low level of incorporation of (R) -β 2 -OH-BocK in cells relative to OH-BocK is due to bottlenecks that occur after tRNA Pyl acylation. One likely bottleneck is EF-Tu, which engages poorly in vitro with tRNA Phe when it is acylated with (R) -or (S) -β 2 -Phe or, notably, with (R) -or (S) -β 3 -Phe ( 11 ), but other bottlenecks cannot be ruled out. Another interesting feature of the acylation of tRNA Pyl with (R)- or (S)- β 2 -OH-BocK in vitro was the appearance of diacylated tRNA products ( 7 ). Diacylated tRNAs–tRNAs acylated on both the 2’ and 3’-hydroxyl groups–were first observed decades ago when T. thermophilus PheRS was used to acylate E. coli tRNA Phe with Phe in vitro ( 12 ). Several years later it was reported that a modified variant of E. coli tRNA Ala chemically diacylated with alanine or allylglycine supported the incorporation of these monomers into protein in an E. coli cell lysate ( 13 ). More recent work detected various diacylated tRNA Pyl species in vitro when tRNA Pyl was reacted with PylRS or variants thereof in the presence of OH-BocK, (R)- and (S) -β 2 -OH-BocK, des-amino BocK, and L-α-amino-, α-thio-, and L-α-hydroxy-Phe ( 7 , 9 ). As far as we know, diacylated tRNAs have never been detected in cells. We applied the PARTI workflow to evaluate whether tRNA Pyl is diacylated in cells expressing PylRS in the presence of OH-BocK, (R)- β 2 -OH-BocK, or (S)- β 2 -OH-BocK. No evidence for tRNA diacylation was observed when the cells were supplemented with either OH-BocK or (S) -β 2 -OH-BocK ( Figure 5D and Supplementary Information, Figure S6, S7 ). Diacylation of tRNA Pyl was detected, however, in cells supplemented with (R) -β 2 -OH-BocK ( Figure 5D and Supplementary Information, Figure S6, S7 ). The di- (R) -β 2 -OH-BocK-Ade detected from RNA isolated from cells was identical in both elution time and exact mass to di- (R) -β 2 -OH-BocK-Ade detected after in vitro tRNA Pyl acylation reactions ( Supplementary Information, Figure S6, S7, S9 ). In vitro , the relative levels of mono- and diacylated tRNA Pyl are related linearly to enzyme concentration ( 7 ). The relative levels of mono- and diacylated tRNA Pyl detected from cells correspond to in vitro acylation reactions performed previously which used less than 2.5 µM PylRS, 10 mM (R) -β 2 -OH-BocK, and 25 µM tRNA Pyl ( 7 ) ( Supplementary Information, Figure S5 ). Although the detected level of diacylated tRNA Pyl in cells is relatively low, we cannot rule out that it does not contribute, at least in part, to the lower yield of intact protein containing (R) -β 2 -OH-BocK. PARTI detects in vivo acylation of monomers that have not yet been reported as ribosome elongation substrates There is great interest in expanding ribosome chemistry beyond simple amine and hydroxyl/thiol nucleophiles to generate ketone products containing a CC bond in place of the canonical CN or CO/CS bonds. Ribosome products containing backbone ketones can be generated by post-translational acyl rearrangements ( 5 ), but have not yet been detected as direct ribosome products. Direct CC bond formation within the ribosome active site demands one or more aaRS enzymes that acylate tRNA with a monomer capable of establishing a proximal carbon-centered nucleophile under physiological conditions. Previous work has shown that PylRS and several variants thereof (FRS1, FRS2, FRSA) acylate tRNA Pyl in vitro with benzylmalonate derivatives capable of generating a carbon-centered nucleophile after decarboxylation, as occurs during reactions catalyzed by polyketide synthases ( 9 , 42 , 43 ). Benzylmalonate derivatives shown to be substrates for the PylRS variant FRSA include meta -trifluoromethyl-2-benzylmalonate ( m -CF 3 -bma), meta -bromo-2-benzylmalonate ( m -Br-bma), and meta -methyl-2-benzylmalonate ( m -CH 3 -bma) ( Figure 6A ) ( 9 ). We used PARTI to establish whether FRSA could acylate tRNA Pyl in cells with each of these benzylmalonate derivatives. Download figure Open in new tab Figure 6: Benzylmalonate derivatives are substrates for FRSA in cells. (A) Structures of meta -bromo-phenylalanine ( m -Br-Phe), meta -bromo-2-benzylmalonate ( m -Br-bma), meta -methyl-2-benzylmalonate ( m -CH 3 -bma) and meta -trifluoromethyl-2-benzylmalonate ( m -CF 3 -bma). (B) Structure and calculated mass of m- Br-Phe-Ade and overlaid EICs of calculated [M+H] for m- Br-Phe-Ade, (at left, [M+H]: 493.0830 Da) normalized to Leu-Enk (at right, [M+H]: 556.2766 Da) after PARTI using RNA isolated from E. coli C321 cells expressing Ma FRSA and Ma tRNA Pyl and grown with (brown) or without (gray) 1 mM m- Br-Phe. (C) Structure and calculated mass of m- CF 3 -bma-Ade and overlaid EICs of calculated [M+H] for m- CF 3 -bma-Ade, (at left, [M+H]: 512.1388 Da) normalized to Leu-Enk (at right, [M+H]: 556.2766 Da) after PARTI with RNA isolated from E. coli C321 cells expressing Ma FRSA and Ma tRNA Pyl and grown with (blue) or without (gray) 1 mM m- CF 3 -bma. (D) Structure and calculated mass of m- Br-bma-Ade and overlaid EICs of calculated [M+H] for m- Br-bma-Ade, (at left, [M+H]: 522.0619 Da) normalized to Leu-Enk (at right, [M+H]: 556.2766 Da) after PARTI with RNA from E. coli C321 cells expressing Ma FRSA and Ma tRNA Pyl and grown with (green) or without (gray) 1 mM m- Br-bma. (E) Structure and calculated mass of m- CH 3 -bma-Ade and overlaid EICs of calculated [M+H] for m- CH 3 -bma-Ade, (at left, [M+H]: 458.1670 Da) and Leu-Enk (at right, [M+H]: 556.2766 Da) after PARTI with RNA from E. coli C321 cells expressing Ma FRSA and Ma tRNA Pyl and grown with (pink) or without (gray) 1 mM m- CH 3 -bma. (F) Shown is a bar graph displaying relative amounts of monoacyl-Ade recovered from E. coli C321 cells grown with 1 mM of the corresponding benzylmalonate or α-amino acid substrate and expressing tRNA Pyl and FRSA. Points shown correspond to biological replicates and error bars represent one standard deviation from the average. Data representing the detection of m -Br-Phe-Ade is shown in orange (mean = 0.64; SD = 0.07; n = 3). Data representing the detection of m -CF 3 -bma, m -Br-bma, and m -CH 3 -bma are shown in blue (mean = 0.23; SD = 0.01; n = 2), green (mean = 0.04; SD = 0.03; n = 3), and pink (mean = 0.03; SD = 0.01; n= 3), respectively. Statistical analysis bars represent the results of a one-way ANOVA. p > 0.05 =ns; p ≤ 0.05 = *; p ≤ 0.01 = **; p ≤ 0.001 = ***. To this end, total RNA was isolated from E. coli C321 cells expressing the Ma FRSA/tRNA Pyl pair and supplemented with 1 mM of either meta -bromo-phenylalanine ( m -Br-Phe) as a positive control or one of the three benzylmalonate substrates, and the tRNA Pyl isolated and analyzed via the standard PARTI workflow. In the case of m -Br-Phe as a substrate, extracting the total ion chromatogram for the mass of m -Br-Phe-Ade generates an EIC with two isobaric peaks which were not observed when the cells were grown in the absence of added substrate ( Figure 6B ). In the case of the three benzylmalonate substrates, three peaks were present in each of the EICs which were absent when substrate was not added during cell growth ( Figure 6C-E , Supplementary Information, Figure S10 ). As many as four isobaric peaks are expected when tRNA is monoacylated with a pro-chiral benzylmalonate derivative, as the reaction generates the expected mixture of 2’ and 3’ products that is each a mixture of two diastereomers ( 9 ). Multiple isobaric peaks in the EIC were also observed when tRNA Pyl was acylated in vitro with these substrates and FRSA ( 9 ). Evaluation of the A norm values resulting from acylation of tRNA Pyl in cells with three benzylmalonate substrates shows a pattern of reactivity that parallels the yields of acylated tRNA Pyl observed in vitro ( 9 ). These A norm values imply that the most reactive benzyl malonate in cells is m -CF 3 -bma, followed by m -Br-bma and then m -CH 3 -bma; the ratio of A norm values for these three substrates in cells was 8:3:1 ( Figure 6F ). These A norm values are 3-, 15-, and 25-fold lower than that observed for the non-canonical α-amino acid which served as a positive control, m- Br-Phe ( Figure 6F ) ( 42 ). Notably, although the relative cellular activities of benzylmalonate substrates implied by A norm values parallels acyl-tRNA Pyl yields observed in vitro ( 9 ), the substrate-dependent differences are greater in cells. In vitro , using 10 mM benzylmalonate, 25 µM tRNA Pyl and 2.5 µM FRSA, the detected ratio of 3-adenylated products was 3:2:1 for m -CF 3 -bma, m -Br-bma and m -CH 3 -bma, respectively ( 9 ). Thus m -CF 3 -bma is a substantially better substrate in cells than expected based on in vitro reactivity. Evidence for cellular metabolism of N -Me BocK There is also great interest in the cellular biosynthesis of N -methylated proteins and peptides, as this modification can alter protein conformation and improve stability, bioavailability, target affinity, and selectivity ( 44 , 45 ). Amide N -Me groups can be installed within peptides prepared synthetically using solid phase methods, or using enzymes, or using the ribosome and chemically acylated tRNAs in vitro and in cell lysate ( 23 , 28 , 30 , 46 – 49 ). However, N -methylated proteins have not yet been prepared in live cells for reasons thought to be associated with poor accommodation of N -Me aminoacyl-tRNA by the ribosome ( 22 , 25 ) and/or low affinity for elongation factor Tu (EF-Tu) ( 29 ). Several aminoacyl synthetases have been reported to acylate tRNA with N -Me amino acids in vitro . For example, N -methyl BocK ( N -Me BocK) is a substrate for Methanosarcina mazei PylRS ( 41 ), and N -methylated phenylalanine analogs are substrates for M. alvus PylRS variants ( 9 ). In alignment with these examples, we found that N -Me BocK is also a substrate for Ma PylRS in vitro . Incubation of 5 µM M. alvus PylRS with 25 µM tRNA Pyl and 10 mM BocK resulted in 29% BocK-tRNA Pyl as determined using intact tRNA LC-MS; when the analogous reaction was performed with N -Me BocK, the yield was 26% ( Supplementary Information, Figure S11 ). Even so, when E. coli Top10 or C321 cells expressing Ma PylRS, Ma tRNA Pyl and a superfolder GFP (sfGFP) plasmid containing a single TAG codon at position 200 (sfGFP-200TAG) were supplemented with N -Me BocK, the only GFP product isolated contained BocK, not N -Me BocK ( Figure 7A and Supplementary Information, S12 ). The yield of purified sfGFP-200TAG containing BocK when cells were supplemented with N -Me BocK (8 mg/L) was more than 20% of the yield isolated when cells were supplemented with the equivalent concentration of BocK (36 mg/L). There was no evidence for BocK contamination in the NMR or high-resolution mass spectrum of the N -Me BocK stock ( Supplementary Information, Figure S13A, B ), nor was BocK-Ade detected from LC-HRMS analysis following RNase A treatment of tRNA Pyl acylated in vitro with Ma PylRS and N -Me BocK ( Supplementary Information, Figure S14A ). Download figure Open in new tab Figure 7: Metabolism of PylRS Substrates in vivo is observable from PARTI with tRNA Pyl . (A) Deconvoluted LC-HRMS spectra of sfGFP-200TAG purified from E. coli Top10 cells supplemented with 1 mM BocK (light green, expected mass: 27,662.3 Da) or 1 mM N-Me BocK (dark green, expected mass: 27,676.3 Da). The observed mass of both proteins is 27,662.3 Da. Signal is normalized to the highest count value within each sample and the trace illustrating the sfGFP-200TAG mass when the growth were supplemented with N -Me BocK is shifted upwards on the y-axis for visibility. (B) Schematic summarizing the PARTI workflow for observing populations of acylation of tRNA Pyl with N -Me BocK (blue) as well as its metabolic product BocK (yellow). Also shown are the structures of the RNase A cleavage products BocK-Ade (expected monoacyl mass: 496.2514 Da) or N -Me BocK-Ade (expected monoacyl mass: 510.26707). (C) Overlaid EICs for detection of Leu-Enk (in gray, [M+H]: 556.2766 Da), BocK-Ade (in gold, [M+H]: 496.2514 Da), and N -Me BocK-Ade (in blue, [M+H]: 510.2671 Da) in a PARTI sample from E. coli Top10 cells expressing Ma PylRS and Ma tRNA Pyl grown with no added substrate. (D) Overlaid EICs for detection of Leu-Enk (in gray, [M+H]: 556.2766 Da), BocK-Ade (in gold, [M+H]: 496.2514 Da), and N -Me BocK-Ade (in blue, [M+H]: 510.2671 Da) in a PARTI sample from E. coli Top10 cells expressing Ma PylRS and Ma tRNA Pyl grown with 1 mM BocK. (E) Overlaid EICs for detection of Leu-Enk (in gray, [M+H]: 556.2766 Da), BocK-Ade (in gold and boxed in muted gold, [M+H]: 496.2514 Da), and N -Me BocK-Ade (in blue and boxed in muted blue, [M+H]: 510.2671 Da) in a PARTI sample from E. coli Top10 cells expressing Ma PylRS and Ma tRNA Pyl grown with 1 mM N -Me BocK. ( F) Shown is a bar graph displaying relative amounts of BocK-Ade (yellow) and N -Me BocK-Ade (blue) recovered from E. coli Top10 cells expressing tRNA Pyl and PylRS and grown with 1 mM indicated monomer(s). PARTI was carried out using o-Pyl and graphed values are respective aa-Ade signals normalized to Leu-Enk signal within each LC-HRMS sample. Points in each bar correspond to biological replicates, and different species detected within a given cell growth condition are from the same biological samples. Error bars are one standard deviation from the average. When cells were supplemented with only 1 mM BocK, only BocK-Ade was detected (mean = 1.79 SD= 0.73 n=3). In cells supplemented with only 1 mM N -Me BocK, both N- Me-BocK Ade (mean = 0.57 SD= 0.34 n=3) and BocK-Ade (mean = 0.25 SD = 0.11 n= 3) were observed. When no substrate was added, neither BocK-Ade nor OH-BocK-Ade were detected. Statistical analysis bars represent the results of a one-way ANOVA. p > 0.05 =ns; p ≤ 0.05 = *; p ≤ 0.01 = **; p ≤ 0.001 = ***. Regardless, to eliminate the possibility that BocK contamination in the N -Me BocK stock could account for the expression of sfGFP-200TAG containing BocK, we grew Top10 and C321 cells expressing Ma PylRS, Ma tRNA Pyl and sfGFP-200TAG in the presence of between 1 pM and 1 mM BocK to determine both the lowest BocK concentration that would yield a detectable level of sfGFP-200TAG containing BocK and the required concentration to replicate the signal observed when 1 mM N- Me BocK had been added ( Supplementary Information, Figure S13C, D ). These results indicate that 1 nM BocK is the lowest BocK concentration that would yield a detectable level of sfGFP-200TAG containing BocK. Furthermore, even a 10% impurity of BocK in the N-Me BocK sample would be insufficient to support the level of incorporation observed in either Top10 or C321 E. coli , and the N-Me BocK monomer is at least 99.9% pure ( Supplementary Information, Figure S13E, F ). As BocK contamination in the N -Me BocK stock cannot account for the observed expression of sfGFP-200TAG containing BocK, we hypothesized that N -Me BocK was metabolized into BocK in cells at one or more points prior to translation. To test this hypothesis, we used the PARTI workflow to probe the acylation state of tRNA Pyl in E. coli expressing the PylRS/tRNA Pyl pair and supplemented with N -Me BocK, and probed specifically for whether tRNA Pyl was acylated with BocK, N -Me BocK, or a mixture of the two ( Figure 7B ). We incubated E. coli Top10 cells expressing the Ma PylRS/tRNA Pyl pair with either 1 mM BocK, 1 mM N -Me BocK, or no substrate, and then subjected each isolated RNA population to the PARTI workflow. No peaks corresponding to the mass of either BocK-Ade or N -Me BocK-Ade were detected when no substrate was used to supplement the cell growths ( Figure 7C and Supplementary Information, Figure S14 ). When cells were supplemented with only BocK, LC-HRMS revealed the anticipated pair of isomeric BocK-Ade peaks ( Figure 7D ). We verified that BocK-Ade captured from an in vivo sample exhibited the same retention times as the products of an in vitro tRNA Pyl acylation reaction ( Supplementary Information, Figure S14 ). No peaks corresponding to the mass of N -Me BocK-Ade were detected from either the in vitro or the in vivo sample ( Figure 7D and Supplementary Information, Figure S14 ). However, when we used the PARTI workflow to analyze total RNA isolated from cells supplemented with only N -Me BocK, we detected clear evidence of both N -Me BocK-Ade and BocK-Ade ( Figure 7E ). N -Me BocK-Ade was detected from the in vivo sample as two isobaric peaks that eluted at the same retention time as the products of the in vitro tRNA Pyl acylation reaction ( Supplementary Information, Figure S14) and were absent when no substrate was added to the cell growths ( Figure 7C and Supplementary Information, Figure S14). BocK-Ade was also detected in the sample isolated from cells supplemented with only N -Me BocK as two peaks with a distinct elution time from N -Me BocK-Ade ( Figure 7E ). Again, BocK-Ade eluted at the same retention time as the products of an in vitro tRNA Pyl acylation reaction ( Supplementary Information, Figure S14) ; these peaks were absent when no substrate was added to the cell growths ( Figure 7C and Supplementary Information, Figure S14 ). The A norm value for BocK-Ade in this sample was 86% lower than from the sample supplemented with 1 mM BocK and was approximately one-half the A norm value due to N -Me-BocK-Ade ( Figure 7F ). We carried out additional LC-HRMS experiments with known amounts of acylated in vitro tRNA Pyl to assess if there were significant differences in the ionization efficiencies of BocK-Ade and N -Me-BocK-Ade. In vitro- purified and transcribed tRNA Pyl was acylated with either BocK or N- Me BocK and the yields of BocK-tRNA Pyl and N- Me BocK-tRNA Pyl , respectively, were determined using intact tRNA LC-MS ( Supplementary Information, Figure S11 ). Aliquots from each reaction were treated with RNase A, doped with Leu-Enk, and analyzed using LC-HRMS to determine A norm values for BocK-Ade or N- Me BocK-Ade. Each A norm value was then divided by the yield of the acylation reaction. We observed that A norm /% acylation was 50% higher when BocK was the substrate ( Supplementary Information, Figure S15 ), suggesting that BocK-Ade ionizes 50% more efficiently than N -Me BocK-Ade. When accounting for this difference, BocK-Ade levels are still only about four times lower than N- Me BocK-Ade levels when cells were supplemented with only N -Me BocK. The level of BocK contamination could not account for the observed level of BocK-Ade, supporting the hypothesis that N -Me-BocK is metabolized into BocK. Because no N -Me BocK is incorporated into protein, BocK appears to entirely outcompete N -Me BocK in the translation steps following acylation. DISCUSSION Here we describe PARTI, a mass spectrometry-based assay that provides a snapshot of the cellular acylation state of a user-defined tRNA. PARTI differs from previously reported cellular tRNA acylation assays in terms of the information it provides. Unlike assays that rely on acyl-tRNA hydrolysis and tRNA amplification ( 15 , 16 , 18 , 50 – 52 ), PARTI provides the exact mass of the acylating species rather than simply whether or not the tRNA has been acylated. In most cases, this exact mass is sufficient to confirm that acylation has occurred with the monomer of interest and not a metabolized derivative or a native α-amino acid. And, unlike assays that rely on acyl-tRNA hydrolysis and mass-guided monomer identification ( 16 , 8 , 19 ), PARTI differentiates between mono- and diacylated tRNA products and thereby does not over-estimate the state of tRNA acylation. Although billions of years of evolution have mitigated diacylation as a concern for native α-amino acids, this side reaction remains a concern for non-α-amino acid monomers, as it is unknown how these unusual tRNA species affect the sophisticated interplay of factors and the ribosome that embody rapid translation. We applied the PARTI workflow here to more deeply scrutinize the multiple steps required to introduce non-α-amino acid monomers into proteins in cells. We focused first on β 2 -hydroxy acid monomers, which can be introduced into protein at two separate positions using the PylRS/tRNA Pyl pair from M. alvus ( 7 ). Recent results show that although both enantiomers of β 2 -hydroxy-boc-lysine (β 2 -OH-BocK) are substrates for M. alvus PylRS in vitro , only the (R) enantiomer is introduced into protein in cells, and with yields 10-fold lower than anticipated based on in vitro tRNA acylation data ( 7 ). Computational results imply that enantioselectivity does not involve the ribosome directly ( 7 ). Using PARTI, we discovered that enantioselectivity is due at least in part to low steady-state levels of tRNA Pyl acylated with (S) -β 2 -OH-BocK but not (R) -β 2 -OH-BocK. (S) -β 2 -OH-BocK may fail to enter cells, be altered by metabolization once it arrives, or it may fail to generate a stable acylated tRNA Pyl upon reaction with PylRS. We also discovered using PARTI that the lower level of incorporation of (R) -β 2 -OH-BocK relative to OH-BocK in cells is likely due to factors that follow tRNA acylation, as the steady state levels of the two acylated tRNAs are comparable. Indeed, recent work has shown that both (R) and (S) enantiomers of β 2 - and β 3 -Phe disrupt ternary complex formation with EF-Tu by at least an order of magnitude in vitro and the complexes that do form ostensibly bypass the proofreading stage of mRNA decoding ( 11 ). This finding emphasizes that for monomers that are not α-amino acids, myriad other translation factors as well as the ribosome itself ( 7 ) must also be considered before heteropolymers containing multiple copies of these unusual substrates, alone or in combination, can be prepared at scale. We note that PARTI was also used here to verify that three different benzyl malonate derivatives are sufficiently cell-permeant to support robust levels of tRNA acylation. This finding demonstrates how PARTI may be used as a screen to establish the relative activities of monomers that are not yet ribosome substrates. The final discovery facilitated by PARTI in this work relates to N -Me-α-amino acids that are of enormous current interest for the development of peptide-derived therapeutics N -Me-α-amino acids can be introduced into peptides in reconstituted in vitro translation mixtures ( 22 – 29 , 53 ) and in S-30 cell extracts ( 30 ), and some are excellent substrates for aaRS enzymes in vitro ( 9 , 41 ). Yet there are no examples in which even a single N -Me-α-amino acid has been introduced into a protein in a cell. PARTI revealed that when E. coli expressing the PylRS/tRNA Pyl pair is supplemented with N -Me-BocK, a notable fraction of the isolated tRNA Pyl carried BocK in addition to the portion acylated with N -Me-BocK. Furthermore, incorporation of only BocK into sfGFP was observed, implying that N -Me-BocK is metabolized via an unknown pathway into BocK in cells. Further work will be required to establish whether this metabolism targets the free amino acid or the acyl-tRNA and whether other N -alkyl amino acids are equally affected. Indeed, it is likely that the efficient incorporation of N -Me backbones in vivo may require strain engineering, as used previously to mitigate the metabolic conversion of α-hydroxy acids into α-amino acids ( 3 , 9 ). In summary, the PARTI assay reported here effectively bridges the informational gap between in vitro acylation and cellular translation. Its ease and simplicity should benefit ongoing efforts to study and improve the cellular incorporation of non-α-amino acid monomers into proteins. DATA AVAILABILITY All data in the manuscript are available in the Supplementary Data, and raw data is available upon request. FUNDING This work was supported by the NSF Center for Genetically Encoded Materials, an NSF Center for Chemical Innovation (C-GEM; CHE 2002182). M.A.P was supported by the Shurl and Kay Curci Foundation. C.K.S. was supported by the Miller Institute for Basic Research in Science, University of California, Berkeley. Conflict of interest statement None declared. ACKNOWLEDGEMENTS The authors are grateful to members of the Schepartz labs for helpful discussion, and especially to Lauren Lesiak, Angel Vázquez Maldonado, and Dr. Daniel Brauer for comments on the manuscript. We also thank Leah Roe for providing benzylmalonate substrates, Noah Hamlish for chemically competent C321 cells, and Alex Solivan for obtaining NMR data. Footnotes Supplementary Figure 5 was misreferenced in the text - the new file has the correct references. REFERENCES 1. ↵ Kwon , D . ( 2023 ) How scientists are hacking the genetic code to give proteins new powers . Nature , 618 , 874 – 876 . OpenUrl CrossRef PubMed 2. ↵ England , P. M. , Zhang , Y. , Dougherty , D. A. and Lester , H. A . ( 1999 ) Backbone mutations in transmembrane domains of a ligand-gated ion channel: implications for the mechanism of gating . Cell , 96 , 89 – 98 . OpenUrl CrossRef PubMed Web of Science 3. ↵ Guo , J. , Wang , J. , Anderson , J. C. and Schultz , P. G . ( 2008 ) Addition of an α-Hydroxy Acid to the Genetic Code of Bacteria . Angew. Chem. Int. Ed ., 47 , 722 – 725 . OpenUrl CrossRef PubMed Web of Science 4. ↵ Roe , L. T. , Schissel , C. K. , Dover , T. L. , Shah , B. , Hamlish , N. X. , Zheng , S. , Dilworth , D. A. , Wong , N. , Zhang , Z. , Chatterjee , A. , et al. ( 2023 ) Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β2-peptide linkages . doi: 10.1101/2023.10.03.560714 . OpenUrl Abstract / FREE Full Text 5. ↵ Schissel , C. , Roberts-Mataric , H. , Garcia , I. , Kang , H. , Francis , M. and Schepartz , A ( 2024 ) Ribosomal Synthesis of Ketone-containing Peptide Backbone via O to C Acyl Shift . doi: 10.26434/chemrxiv-2024-bkzp3 . OpenUrl CrossRef 6. ↵ Melo Czekster , C. , Robertson , W. E. , Walker , A. S. , Söll , D. and Schepartz , A. ( 2016 ) In Vivo Biosynthesis of a β-Amino Acid-Containing Protein . J. Am. Chem. Soc ., 138 , 5194 – 5197 . OpenUrl CrossRef PubMed 7. ↵ Hamlish , N. X. , Abramyan , A. M. , Shah , B. , Zhang , Z. and Schepartz , A . ( 2024 ) Incorporation of Multiple β2-Hydroxy Acids into a Protein In Vivo Using an Orthogonal Aminoacyl-tRNA Synthetase . ACS Cent. Sci ., 10 , 1044 – 1053 . OpenUrl CrossRef PubMed 8. ↵ Dunkelmann , D. L. , Piedrafita , C. , Dickson , A. , Liu , K. C. , Elliott , T. S. , Fiedler , M. , Bellini , D. , Zhou , A. , Cervettini , D. and Chin , J. W . ( 2024 ) Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism . Nature , 625 , 603 – 610 . OpenUrl CrossRef PubMed 9. ↵ Fricke , R. , Swenson , C. V. , Roe , L. T. , Hamlish , N. X. , Shah , B. , Zhang , Z. , Ficaretta , E. , Ad , O. , Smaga , S. , Gee , C. L. , et al. ( 2023 ) Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo . Nat. Chem ., 15 , 960 – 971 . OpenUrl CrossRef PubMed 10. Watson , Z. L. , Knudson , I. J. , Ward , F. R. , Miller , S. J. , Cate , J. H. D. , Schepartz , A. and Abramyan , A. M . ( 2023 ) Atomistic simulations of the Escherichia coli ribosome provide selection criteria for translationally active substrates . Nat. Chem ., 15 , 913 – 921 . OpenUrl CrossRef PubMed 11. ↵ Cruz-Navarrete , F. A. , Griffin , W. C. , Chan , Y.-C. , Martin , M. I. , Alejo , J. L. , Natchiar , S. K. , Knudson , I. J. , Altman , R. B. , Schepartz , A. , Miller , S. J. , et al. ( 2024 ) β-amino acids reduce ternary complex stability and alter the translation elongation mechanism . doi: 10.1101/2024.02.24.581891 . OpenUrl Abstract / FREE Full Text 12. ↵ Stepanov , V. G. , Moor , N. A. , Ankilova , V. N. and Lavrik , O. I . ( 1992 ) Phenylalanyl-tRNA synthetase from Thermus thermophilus can attach two molecules of phenylalanine to tRNAPhe . FEBS Lett ., 311 , 192 – 194 . OpenUrl CrossRef PubMed Web of Science 13. ↵ Wang , B. , Zhou , J. , Lodder , M. , Anderson , R. D. and Hecht , S. M . ( 2006 ) Tandemly Activated tRNAs as Participants in Protein Synthesis* . J. Biol. Chem ., 281 , 13865 – 13868 . OpenUrl Abstract / FREE Full Text 14. ↵ Spinck , M. , Piedrafita , C. , Robertson , W. E. , Elliott , T. S. , Cervettini , D. , de la Torre , D . and Chin , J. W. ( 2023 ) Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles . Nat. Chem ., 15 , 61 – 69 . OpenUrl CrossRef PubMed 15. ↵ Gaston , K. W. , Rubio , M. A. T. and Alfonzo , J. D . ( 2008 ) OXOPAP assay: for selective amplification of aminoacylated tRNAs from total cellular fractions . Methods San Diego Calif , 44 , 170 – 175 . OpenUrl CrossRef PubMed Web of Science 16. ↵ Cervettini , D. , Tang , S. , Fried , S. D. , Willis , J. C. W. , Funke , L. F. H. , Colwell , L. J. and Chin , J. W . ( 2020 ) Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase–tRNA pairs . Nat. Biotechnol ., 38 , 989 – 999 . OpenUrl CrossRef PubMed 17. Watkins , R. R. , Kavoor , A. and Musier-Forsyth , K . ( 2024 ) Strategies for Detecting Aminoacylation and Aminoacyl-tRNA Editing in vitro and in Cells . Isr. J. Chem ., 64 , e202400009 . OpenUrl 18. ↵ Soni , C. , Prywes , N. , Hall , M. , Nair , M. A. , Savage , D. F. , Schepartz , A. and Chatterjee , A . ( 2024 ) A Translation-Independent Directed Evolution Strategy to Engineer Aminoacyl-tRNA Synthetases . ACS Cent. Sci ., 10 , 1211 – 1220 . OpenUrl CrossRef PubMed 19. ↵ Mohler , K. , Mann , R. and Ibba , M . ( 2017 ) Isoacceptor specific characterization of tRNA aminoacylation and misacylation in vivo . Methods , 113 , 127 – 131 . OpenUrl CrossRef PubMed 20. ↵ Dunkelmann , D. L. , Piedrafita , C. , Dickson , A. , Liu , K. C. , Elliott , T. S. , Fiedler , M. , Bellini , D. , Zhou , A. , Cervettini , D. and Chin , J. W . ( 2024 ) Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism . Nature , 625 , 603 – 610 . OpenUrl CrossRef PubMed 21. ↵ Ad , O. , Hoffman , K. S. , Cairns , A. G. , Featherston , A. L. , Miller , S. J. , Söll , D. and Schepartz , A . ( 2019 ) Translation of Diverse Aramid- and 1,3-Dicarbonyl-peptides by Wild Type Ribosomes in Vitro . ACS Cent. Sci ., 5 , 1289 – 1294 . OpenUrl CrossRef PubMed 22. ↵ Zhang , B. , Tan , Z. , Dickson , L. G. , Nalam , M. N. L. , Cornish , V. W. and Forster , A. C . ( 2007 ) Specificity of Translation for N-Alkyl Amino Acids . J. Am. Chem. Soc ., 129 , 11316 – 11317 . OpenUrl CrossRef PubMed Web of Science 23. ↵ Kawakami , T. , Murakami , H. and Suga , H . ( 2008 ) Messenger RNA-programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides . Chem. Biol ., 15 , 32 – 42 . OpenUrl CrossRef PubMed Web of Science 24. Subtelny , A. O. , Hartman , M. C. T. and Szostak , J. W . ( 2008 ) Ribosomal Synthesis of N-Methyl Peptides . J. Am. Chem. Soc ., 130 , 6131 – 6136 . OpenUrl CrossRef PubMed Web of Science 25. ↵ Pavlov , M. Y. , Watts , R. E. , Tan , Z. , Cornish , V. W. , Ehrenberg , M. and Forster , A. C . ( 2008 ) Slow peptide bond formation by proline and other N-alkylamino acids in translation . Proc. Natl. Acad. Sci. U. S. A ., 106 , 50 . OpenUrl PubMed 26. Kawakami , T. , Murakami , H. and Suga , H . ( 2008 ) Ribosomal Synthesis of Polypeptoids and Peptoid−Peptide Hybrids . J. Am. Chem. Soc ., 130 , 16861 – 16863 . OpenUrl CrossRef PubMed Web of Science 27. Subtelny , A. O. , Hartman , M. C. T. and Szostak , J. W . ( 2011 ) Optimal codon choice can improve the efficiency and fidelity of N-methyl amino acid incorporation into peptides by in-vitro translation . Angew. Chem. Int. Ed Engl ., 50 , 3164 – 3167 . OpenUrl CrossRef PubMed 28. ↵ Wang , J. , Kwiatkowski , M. , Pavlov , M. Y. , Ehrenberg , M. and Forster , A. C . ( 2014 ) Peptide Formation by N-Methyl Amino Acids in Translation Is Hastened by Higher pH and tRNAPro . ACS Chem. Biol ., 9 , 1303 – 1311 . OpenUrl CrossRef PubMed 29. ↵ Iwane , Y. , Kimura , H. , Katoh , T. and Suga , H . ( 2021 ) Uniform affinity-tuning of N-methyl-aminoacyl-tRNAs to EF-Tu enhances their multiple incorporation . Nucleic Acids Res ., 49 , 10807 – 10817 . OpenUrl CrossRef PubMed 30. ↵ Zhang , C. , Chen , S. , Fu , X. , Dedkova , L. M. and Hecht , S. M . ( 2024 ) Enhancement of N-Methyl Amino Acid Incorporation into Proteins and Peptides Using Modified Bacterial Ribosomes and Elongation Factor P . ACS Chem. Biol ., 19 , 1330 – 1338 . OpenUrl CrossRef PubMed 31. ↵ Tangpradabkul , T. , Palo , M. , Townley , J. , Hsu , K. B. , participants , E. , Smaga , S. , Das , R. and Schepartz , A . ( 2024 ) Minimization of the E. coli ribosome, aided and optimized by community science . Nucleic Acids Res ., 52 , 1027 – 1042 . OpenUrl CrossRef PubMed 32. ↵ Fricke , R. , Knudson , I. and Schepartz , A. ( 2023 ) Direct, quantitative, and comprehensive analysis of tRNA acylation using intact tRNA liquid-chromatography mass-spectrometry . doi: 10.1101/2023.07.14.549096 . OpenUrl Abstract / FREE Full Text 33. ↵ Knox , S. L. , Wissner , R. , Piszkiewicz , S. and Schepartz , A . ( 2021 ) Cytosolic Delivery of Argininosuccinate Synthetase Using a Cell-Permeant Miniature Protein . ACS Cent. Sci ., 7 , 641 – 649 . OpenUrl CrossRef PubMed 34. ↵ Polycarpo , C. , Ambrogelly , A. , Bérubé , A. , Winbush , S. M. , McCloskey , J. A. , Crain , P. F. , Wood , J. L. and Söll , D . ( 2004 ) An aminoacyl-tRNA synthetase that specifically activates pyrrolysine . Proc. Natl. Acad. Sci. U. S. A ., 101 , 12450 – 12454 . OpenUrl Abstract / FREE Full Text 35. ↵ Lajoie , M. J. , Rovner , A. J. , Goodman , D. B. , Aerni , H.-R. , Haimovich , A. D. , Kuznetsov , G. , Mercer , J. A. , Wang , H. H. , Carr , P. A. , Mosberg , J. A. , et al. ( 2013 ) Genomically Recoded Organisms Expand Biological Functions . Science , 342 , 357 – 360 . OpenUrl Abstract / FREE Full Text 36. ↵ Kesik-Brodacka , M. , Romanik , A. , Mikiewicz-Sygula , D. , Plucienniczak , G. and Plucienniczak , A . ( 2012 ) A novel system for stable, high-level expression from the T7 promoter . Microb. Cell Factories , 11 , 109 . OpenUrl CrossRef 37. ↵ Wei , B. , Gunzner-Toste , J. , Yao , H. , Wang , T. , Wang , J. , Xu , Z. , Chen , J. , Wai , J. , Nonomiya , J. , Tsai , S. P. , et al. ( 2018 ) Discovery of Peptidomimetic Antibody-Drug Conjugate Linkers with Enhanced Protease Specificity . J. Med. Chem ., 61 , 989 – 1000 . OpenUrl CrossRef PubMed 38. ↵ Pittard , J. , Praszkier , J. , Certoma , A. , Eggertsson , G. , Gowrishankar , J. , Narasaiah , G. and Whipp , M. J . ( 1990 ) Evidence that there are only two tRNA(Phe) genes in Escherichia coli . J. Bacteriol ., 172 , 6077 – 6083 . OpenUrl Abstract / FREE Full Text 39. ↵ Yegian , C. D. , Stent , G. S. and Martin , E. M . ( 1966 ) Intracellular condition of Escherichia coli transfer RNA . Proc. Natl. Acad. Sci. U. S. A ., 55 , 839 – 846 . OpenUrl FREE Full Text 40. ↵ Sztáray , J. , Memboeuf , A. , Drahos , L. and Vékey , K . ( 2011 ) Leucine enkephalin--a mass spectrometry standard . Mass Spectrom. Rev ., 30 , 298 – 320 . OpenUrl CrossRef PubMed 41. ↵ Kobayashi , T. , Yanagisawa , T. , Sakamoto , K. and Yokoyama , S . ( 2009 ) Recognition of Non-α-amino Substrates by Pyrrolysyl-tRNA Synthetase . J. Mol. Biol ., 385 , 1352 – 1360 . OpenUrl CrossRef PubMed Web of Science 42. ↵ Wang , Y. , Russell , W. , Wang , Z. , Wan , W. , Dodd , L. , Pai , P. , Russell , D. and Liu , W . ( 2011 ) The de novo engineering of pyrrolysyl-tRNA synthetase for genetic incorporation of L-phenylalanine and its derivatives . Mol. Biosyst ., 7 , 714 – 717 . OpenUrl CrossRef PubMed 43. ↵ Soohoo , A. M. , Cogan , D. P. , Brodsky , K. L. and Khosla , C . ( 2024 ) Structure and Mechanisms of Assembly-Line Polyketide Synthases . Annu. Rev. Biochem ., 93 , 471 – 498 . OpenUrl CrossRef PubMed 44. ↵ Fiacco , S. V. and Roberts , R. W . ( 2008 ) N-Methyl Scanning Mutagenesis Generates Protease-Resistant G Protein Ligands with Improved Affinity and Selectivity. Chembiochem Eur . J. Chem. Biol ., 9 , 2200 – 2203 . OpenUrl 45. ↵ Schwochert , J. , Turner , R. , Thang , M. , Berkeley , R. F. , Ponkey , A. R. , Rodriguez , K. M. , Leung , S. S. F. , Khunte , B. , Goetz , G. , Limberakis , C. , et al. ( 2015 ) Peptide to Peptoid Substitutions Increase Cell Permeability in Cyclic Hexapeptides . Org. Lett ., 17 , 2928 – 2931 . OpenUrl CrossRef PubMed 46. ↵ Chatterjee , J. , Gilon , C. , Hoffman , A. and Kessler , H . ( 2008 ) N-Methylation of Peptides: A New Perspective in Medicinal Chemistry . Acc. Chem. Res ., 41 , 1331 – 1342 . OpenUrl CrossRef PubMed Web of Science 47. Ude , S. , Lassak , J. , Starosta , A. L. , Kraxenberger , T. , Wilson , D. N. and Jung , K . ( 2013 ) Translation Elongation Factor EF-P Alleviates Ribosome Stalling at Polyproline Stretches . Science , 339 , 82 – 85 . OpenUrl Abstract / FREE Full Text 48. van der Velden , N. S. , Kälin , N. , Helf , M. J. , Piel , J. , Freeman , M. F. and Künzler , M. ( 2017 ) Autocatalytic backbone N-methylation in a family of ribosomal peptide natural products . Nat. Chem. Biol ., 13 , 833 – 835 . OpenUrl CrossRef PubMed 49. ↵ Ramm , S. , Krawczyk , B. , Mühlenweg , A. , Poch , A. , Mösker , E. and Süssmuth , R. D . ( 2017 ) A Self-Sacrificing N-Methyltransferase Is the Precursor of the Fungal Natural Product Omphalotin . Angew. Chem. Int. Ed ., 56 , 9994 – 9997 . OpenUrl CrossRef 50. ↵ Dyer , J. R . ( 1956 ) Use of periodate oxidations in biochemical analysis . Methods Biochem. Anal ., 3 , 111 – 152 . OpenUrl CrossRef PubMed 51. Tsukamoto , Y. , Nakamura , Y. , Hirata , M. , Sakate , R. and Kimura , T . ( 2022 ) i-tRAP (individual tRNA acylation PCR): A convenient method for selective quantification of tRNA charging . RNA N. Y. N , 29 , 111 – 122 . OpenUrl 52. ↵ Davidsen , K. and Sullivan , L. B . ( 2024 ) A robust method for measuring aminoacylation through tRNA-Seq . bioRxiv , doi: 10.1101/2023.07.31.551363 . OpenUrl Abstract / FREE Full Text 53. ↵ Katoh , T. and Suga , H . ( 2023 ) Ribosomal incorporation of negatively charged d-α- and N-methyl-l-α-amino acids enhanced by EF-Sep . Philos. Trans. R. Soc. Lond. B. Biol. Sci ., 378 , 20220038. View the discussion thread. Back to top Previous Next Posted January 16, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Monitoring monomer-specific acyl-tRNA levels in cells with PARTI Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Monitoring monomer-specific acyl-tRNA levels in cells with PARTI Meghan A. Pressimone , Carly K. Schissel , Isabella H. Goss , Cameron V. Swenson , Alanna Schepartz bioRxiv 2025.01.14.633079; doi: https://doi.org/10.1101/2025.01.14.633079 Share This Article: Copy Citation Tools Monitoring monomer-specific acyl-tRNA levels in cells with PARTI Meghan A. Pressimone , Carly K. Schissel , Isabella H. Goss , Cameron V. Swenson , Alanna Schepartz bioRxiv 2025.01.14.633079; doi: https://doi.org/10.1101/2025.01.14.633079 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biochemistry Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17690) Bioengineering (13892) Bioinformatics (41935) Biophysics (21451) Cancer Biology (18587) Cell Biology (25499) Clinical Trials (138) Developmental Biology (13377) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24318) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88601) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15152) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)