TCEP-Mediated Protein Hydrolysis: Highly Selective Cleavage C-terminal to Aspartic Acid

TCEP-Mediated Protein Hydrolysis: Highly Selective Cleavage C-terminal to Aspartic Acid | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 29 May 2025 V1 Latest version Share on TCEP-Mediated Protein Hydrolysis: Highly Selective Cleavage C-terminal to Aspartic Acid Authors : Daniel S. McCracken 0000-0001-8759-3539 [email protected] , David J. Foreman , Kyle G. Esposito , and Feras Hatahet Authors Info & Affiliations https://doi.org/10.22541/au.174849500.05592731/v1 Published Rapid Communications in Mass Spectrometry Version of record Peer review timeline 731 views 265 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Tris(2-carboxyethyl)phosphine (TCEP) is a commonly used laboratory reagent for the purposes of reducing disulfide bonds. TCEP is often preferred over alternative reducing reagents, such as dithiothreitol (DTT), due its strong redox potential, effectiveness in a broad pH range, and handling considerations. Despite this, many side reactions involving TCEP have been poorly categorized and understood. Utilizing a combination of gel electrophoresis and mass spectrometry techniques, we have discovered a unique and novel mechanism by which TCEP engages in the hydrolysis of proteins. This behavior has been observed to be site specific to the carboxyl terminus of aspartic acid residues. Cleavage at this location is completely independent of the reduction of disulfide bonds in proteins due to the observance of this phenomenon in proteins lacking disulfide bonds, and some lacking cysteines altogether. While the specific chemistry of this hydrolysis is unknown, this discovery has potentially wide-ranging impacts as a sample preparation tool for mass spectrometry analysis, as well as being cautionary towards other common laboratory uses of this ubiquitous reagent where it could potentially cause unwanted hydrolysis of proteins being studied. TCEP-Mediated Protein Hydrolysis: Highly Selective Cleavage C-terminal to Aspartic Acid Daniel S. McCracken 1‡* , David J. Foreman 1‡ , Kyle G. Esposito 1 , Feras Hatahet 1 1 Analytical Research & Development, Merck & Co., Inc., 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States ‡D.S.M. and D.J.F. contributed equally to this work. ABSTRACT: Tris(2-carboxyethyl)phosphine (TCEP) is a commonly used laboratory reagent for the purposes of reducing disulfide bonds. TCEP is often preferred over alternative reducing reagents, such as dithiothreitol (DTT), due its strong redox potential, effectiveness in a broad pH range, and handling considerations. Despite this, many side reactions involving TCEP have been poorly categorized and understood. Utilizing a combination of gel electrophoresis and mass spectrometry techniques, we have discovered a unique and novel mechanism by which TCEP engages in the hydrolysis of proteins. This behavior has been observed to be site specific to the carboxyl terminus of aspartic acid residues. Cleavage at this location is completely independent of the reduction of disulfide bonds in proteins due to the observance of this phenomenon in proteins lacking disulfide bonds, and some lacking cysteines altogether. While the specific chemistry of this hydrolysis is unknown, this discovery has potentially wide-ranging impacts as a sample preparation tool for mass spectrometry analysis, as well as being cautionary towards other common laboratory uses of this ubiquitous reagent where it could potentially cause unwanted hydrolysis of proteins being studied. INTRODUCTION Tris(2-carboxyethyl)phosphine (TCEP) is extensively used for the principle purpose of reducing disulfide bonds1. It is often preferred over other common reductants such as dithiothreitol (DTT) and beta-mercapthoethanol (BME) due to its strong redox potential, effectiveness over a wide pH range, stability, and odorlessness 1, 2 . Several thorough comparisons have already been performed between reducing agents which show TCEP to be superior in many assays to use of DTT 2-4 . The structures of TCEP, DTT, and BME are provided in Figure 1A. The dissimilarity between TCEP and other common reductants, including the lack of thiols, is notable. DTT and BME function by utilizing thiol-disulfide exchange reactions to transfer the disulfide bonds from proteins to themselves. TCEP, in contrast, utilizes a nucleophilic attack from the phosphate atom in the phosphine to one of the sulfurs in the disulfide, and then oxidizes with water to release the thiol 5 . This mechanism is illustrated in Figure 1B. Unlike DTT, TCEP is not itself a thiol, and is generally regarded as inert towards the functional groups present in amino acid residues. Therefore, elimination of TCEP is often not as critical as the clearance of DTT for potential downstream assays 6 . However, a number of side reactions of TCEP have been investigated, including, but not limited to, peptide cleavage at azide homoalanine 7 , the breaking of carbon bonds in cysteine 1 , and para-azidobenzyl cleavage for use in bioconjugation 8 . These investigations establish that TCEP could potentially be a useful laboratory reagent for applications outside of disulfide bond reduction. Numerous other common laboratory reagents have found applications in non-enzymatic proteolysis. For example, acid hydrolysis involves heating proteins in strong acids, commonly hydrochloric acid, for prolonged periods of time 9-11 . The result is the non-specific hydrolysis of protein amide bonds. Similarly, Edman’s degradation results in the N-terminal cleavage of peptide bonds 12 . Cyanogen bromide, on the other hand, offers site specific hydrolysis at methionine residues 13-15 . Similar chemical cleavage protocols have been reported for site specific cleavage at tryptophan 16-18 , aspartic acid 19, 20 , serine 21 , cysteine 7, 22 , and asparagine residues23. Site specific proteolysis is a cornerstone of bottom-up mass spectrometry workflows. In bottom-up approaches, proteins are digested, and the resulting peptides are separated with liquid chromatography (LC) and analyzed via tandem mass spectrometry (MS/MS) 24, 25 . These workflows can reveal information about the identity 26 and the quantity 27, 28 of the proteins present within the sample. Trypsin is often regarded as the enzyme of choice for bottom-up applications due to its high specificity, availability, affordability, and ease of use 29-31 . However, digestion with alternative proteases affords complementary sequence information and can lead to post- Figure 1. A) The commonly used reductants of tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), and beta-mercaptoethanol (BME). B) Mechanism of action of TCEP. TCEP engages in nucleophilic attack of a disulfide bonded thiol, and then oxidizes with water to reduce disulfide bonds. translational modification (PTM) localization and improved quantification 31-37 . Chemically based cleavage of amide bonds typically lacks specificity or requires exceptionally harsh conditions 7, 22, 38 . Here, we report the highly specific cleavage of proteins C-terminal to aspartic acid cleavage of proteins in the presence of TCEP. It is demonstrated that this phenomenon is highly dependent on the concentration of TCEP, the incubation temperature, and the incubation time. The purpose of this work is twofold. First, it demonstrates that TCEP is a viable alternative for site-specific protein hydrolysis. Additionally, it serves as a cautionary tale for experiments involving the use of TCEP since this phenomenon was observed with TCEP concentrations and incubation temperatures recommended by the manufacturer. Experimental Procedures/ materials and methods/ methods SDS-PAGE 4x Sample Buffer of 0.25 M Tris pH 7.0, 8% SDS, 40% Glycerol, 0.008% Bromophenol Blue was prepared from Trizma hydrocloride solution (Sigma Aldrich, St. Louis, MO, USA), sodium n-dodecyl sulfate (Alfa Aesar, Tewksbury, MA, USA), glycerol (Fisher Chemical, Waltham, MA, USA), bromophenol blue (Bio-Rad, Hercules, CA, USA). The concentration of recombinant L1 was measured by UV/Vis. Bovine serum albumin (BSA) (Sigma-Adrich, St. Louis, MO, USA) and lysozyme (Sigma Aldrich, St. Louis, MO, USA) concentrations are from commercial stocks. Proteins were diluted to 0.32 µg/µL for loading into Criterion 26 well 4-20% tris-HCl gels (Bio-Rad, Hercules, CA, USA) with 4x sample buffer with TCEP (Thermo Scientific, Waltham, MA, USA) and/or DTT (Thermo Scientific, Waltham, MA, USA), and incubated as described in results and discussion, 12.5 µL was loaded into each lane, and run at 250 V for 35 minutes in tris-glycine running buffer (Invitrogen, Waltham, MA, USA). Gels were rinsed with Milli-Q water for 5 minutes, then fixed with 12% TCA (Ricca Chemical, Arlington, TX, USA) for 1 hour. Gels were deacidified with six 10-minute rinses with Milli-Q water and were subsequently stained overnight with GelCode Blue (Thermo Scientific, Waltham, MA, USA). They were then destained with six 1-hour rinses with Milli-Q water, and imaged using the Bio-Rad Gel Doc (Bio-Rad, Hercules, CA, USA), auto-optimized with the Image Lab software (Bio-Rad, Hercules, CA, USA) for faint bands. Mass Spectrometry of Recombinant L1 For experiments involving recombinant L1, two 500 µL aliquots were concentrated via Amicon Ultra – 0.5 mL, 10,000 MWCO centrifugal filters (MilliporeSigma, Burlington, MA, USA). The retained sample was combined. A total of three samples were prepared by diluting 15 µL of the concentrated material with 8.0 M guanidine HCl (Thermo Scientific, Waltham, MA, USA) and reductant, either 500 mM TCEP (Alfa Aesar, Tewksbury, MA, USA) or 500 mM DTT (Thermo Scientific, Waltham, MA, USA). The sample containing 50 mM TCEP, 6.0 M guanidine HCl was incubated at 80 o C for 2 minutes, the sample containing 90 mM TCEP, 5.5 M guanidine HCl was incubated at 80 o C for 20 minutes, and the sample containing 90 mM DTT, 5.5 M guanidine HCl was incubated at 37 o C for 60 minutes. Samples were analyzed via RP-LC/MS with an Aquity UPLC (Waters, Milford, MA, USA) equipped with a binary solvent manager, column manager, and fluorescence detector coupled to a Vion IMS QTof (Waters, Milford, MA, USA) operating in the High Definition MS mode. Separation was performed using a BIOshell TM IgG 1000Å C4, 2.7 µm, 2.1 x 100 mm (Millipore Sigma, Burlington, MA, USA). The UPLC method consisted of a linear gradient from 38% mobile phase B to 46% mobile phase B over 26 minutes (mobile phase A: 0.15% difluroracetic acid in water (Waters, Milford, MA, USA), mobile phase B: 0.15% difluoracetic acid in acetonitrile) at a flow rate of 0.3 mL/min, a fluorescence excitation wavelength of 280 nm, and a fluorescence emission wavelength of 350 nm. The column temperature was set to 80 o C. Deconvolution was performed using UNIFI (Waters, Milford, MA, USA). Deconvoluted masses were then searched against theoretical hydrolysis products using a program written in MATLAB (MathWorks, Natick, MA, USA). Figure 2. A) Cleavage of recombinant L1, BSA, and lysozyme with varying concentrations of TCEP. The concentration of TCEP is 0mM in lane 1 (left most lane), 5mM in lane 2, 10mM in lane 3, 25mM in lane 4, 50mM in lane 5, and 100mM in lane 6 (right most lane). Samples were incubated for 10 minutes at 70 o C. Asterisk denotes higher order species of recombinant L1. B) Cleavage of recombinant L1, BSA, and lysozyme at various temperatures. The temperature is room temperature in lane 1 (left most lane), 30 o C in lane 2, 40 o C in lane 3, 50 o C in lane 4, 60 o C in lane 5, 70 o C in lane 6, and 80 o C in lane 7 (right most lane). Samples were incubated for 10 minutes with 50mM TCEP. Asterisk denotes higher order species of recombinant L1. Peptide Mapping of Protein Standards For all other mass spectrometry experiments, 1 mg/mL stocks of BSA (Sigma Aldrich, St. Louis, MO, USA), lysozyme (Sigma Aldrich, St. Louis, MO, USA), carbonic anhydrase (Sigma Aldrich, St. Louis, MO, USA), alcohol dehydrogenase (Sigma Aldrich, St. Louis, MO, USA), phosphorylase b (Sigma Aldrich, St. Louis, MO, USA), myoglobin (Sigma Aldrich, St. Louis, MO, USA), and Protein A (Sigma Aldrich, St. Louis, MO, USA) were prepared. For incubation with TCEP, 5 µL of protein stock was combined with 75 µL of 8.0 M guanidine HCl, and 20 µL of 500 mM TCEP. The final solution contained 50 µg of protein, 100 mM TCEP, and 6.0 M guanidine HCl. Samples were incubated overnight at 80 o C. For digestion with trypsin (Promega, Madison, WI, USA), 5 µL of protein stock was combined with 75 µL of 8.0 M guanidine HCl and 20 µL of Optima LC/MS grade water (Fisher Chemical, Waltham, MA, USA). 5 µL of freshly prepared 100 mM DTT (Thermo Scientific, Waltham, MA, USA) was added to each sample, and the samples were incubated at 37 o C for 30 minutes. The solutions were cooled to room temperature and centrifuged briefly. Iodoacetamide (Thermo Scientific, Waltham, MA, USA) was added to a final concentration of 14 mM, from a freshly prepared 100 mM stock solution. Samples were incubated in the dark at 25 o C for 30 minutes. Unreacted iodoacetamide was quenched with the addition of another 5 µL of freshly prepared 100 mM DTT. Samples were incubated in the dark at 25 o C for 15 minutes. Samples were buffer exchanged into 50 mM ammonium bicarbonate (Fluka Analytical, ) using Zeba Spin Desalting Columns (Thermo Scientific, Waltham, MA, USA) per the manufacturer’s instructions. 10 µL of 0.1 µg/µL trypsin (1 µg) (Promega, Madison, WI, USA) was added to each sample. Samples were incubated overnight at 37 o C. The digestion was quenched with the addition of 1.3 µL of 10% formic acid (Fisher Chemical, Waltham, MA, USA). Samples were analyzed via RP-LC/MS/MS with a nanoAquity UPLC (Waters, Milford, MA, USA) equipped with a binary solvent manager and column manager coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific, Waltham, MA, USA) operating in DDA acquisition mode. Separation was performed using an easy EASY-Spray C18, 3 µm, 75 µm x 150 mm (Thermo Scientific, Waltham, MA, USA). The nano LC method consisted of a linear gradient from 0.5% mobile phase B to 40% mobile phase B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile) at a flow rate of 0.3 µL/min and a column temperature of 30 o C. All data were analyzed in Expressionist MS Refiner with (Genedata Inc., Lexington, MA, USA). Details surrounding the data processing workflow are provided in the Supporting Information. Results and Discussion Concentration, Temperature, Time, and pH Dependence of TCEP Mediated Proteolysis as Revealed by SDS-PAGE Recombinant L1, BSA, and lysozyme were incubated with TCEP and analyzed via SDS-PAGE under a variety of conditions to assess the specifics of TCEP mediated proteolytic cleavage. These specific proteins were used because of the first observance of this phenomenon in unrelated recombinant L1 experiments (data not shown), as well as the desire to show relevance in commonly used laboratory proteins. The concentration of TCEP and the incubation temperature were varied during the denaturation and reduction step of SDS-PAGE analysis (Figure 2). Figure 2A illustrates a concentration dependence for proteolytic cleavage. TCEP concentrations were increased from 0 mM to 100 mM while maintaining a constant time (10 minutes) and temperature (70 o C) for incubation. This dependence is readily observed by the increase of low molecular weight band intensity with higher concentrations of TCEP. The effect is more difficult to observe with lysozyme, yet faint bands are still observed with 100 mM TCEP. Figure 2B illustrates a temperature dependence for TCEP mediated cleavage. Here, a constant TCEP concentration (50 mM) was maintained while varying the temperature from room temperature to 80 o C. Incomplete reduction of recombinant L1 at temperatures below 50 o C is observed, as evidenced by the dimer and trimer species at approximately 110 kDa and 165 kDa, respectively. An increase in proteolytic cleavage is observed at temperatures above 50 o C, as shown by the increase in the number of low molecular weight bands and an increase in intensity of these bands. Like recombinant L1, minimal cleavage is observed in BSA below 50 o C and the extent of cleavage increases with temperatures above 50 o C. While the intensity of the low molecular weight bands appears to increase with Figure 3. A) Cleavage of recombinant L1 at various times ranging from 2 minutes to 20 minutes. B) Cleavage of BSA at various times ranging from 2 minutes to 20 minutes. All samples were incubated at 70 o C with 50mM TCEP. reduction of BSA, later we show this phenomenon with proteins lacking disulfide bonds or cysteines. Again, this effect is more difficult to observe with lysozyme, however, proteolytic cleavage increases at higher temperatures. Similarly, an examination of the effect of incubation time reveals increased proteolytic cleavage with longer incubation times (Figure 3). Recombinant L1 and BSA were incubated with 50 mM TCEP at 70 o C. When altering the incubation time between 2 and 20 minutes, in 2-minute increments, cleavage of recombinant L1 (Figure 3A) and BSA (Figure 3B) both increase in a time dependent manner. As previously mentioned, this phenomenon is difficult to visualize in lysozyme and, therefore, lysozyme was not utilized for the time dependent studies of TCEP mediated proteolytic cleavage. The breaking of peptide bonds at aspartic acid residues in dilute acids has been previously reported 19, 20 . To investigate the effect of pH on TCEP mediated proteolysis, the pH of the sample buffer was adjusted with hydrochloric acid and sodium hydroxide. TCEP and protein were both added to a final concentration of 25 mM to minimize any effects on the overall pH of the solution. Samples were incubated at 70 o C for 10 minutes. Figure 4 reveals that the cleavage increases slightly with the alkalinity of the sample buffer, with a marked increase in cleavage at pH 8. This is counter to what is known about acidic cleavage of peptides, where they can be cleaved C-terminal to aspartic acid in dilute acids. This indicates that the TCEP-mediated cleavage has a degree of pH dependance. Figure 4. A) Cleavage of recombinant L1 at various pH. B) Cleavage of BSA at various pH. All samples were incubated at 70 o C with 25mM TCEP for 10 minutes. The pH examined here were pH 4 (lane 1, left most lane), pH 5 (lane 2), pH 6 (lane 3), pH 6.5 (lane 4), pH 7 (lane 5), pH 7.5 (lane 6), and pH 8 (lane 7, right most lane). The results of the SDS-PAGE experiments point towards an interesting and novel mechanism of proteolytic cleavage. Data suggests that this proteolysis occurs at specific cleavage sites, indicated by the presence of distinct bands, rather than as streaking, when the gels are stained. Proteolysis occurs in a concentration, temperature, time, and, to a lesser extent, pH dependent manner. The concentration dependence on TCEP is a strong indicator that TCEP is responsible for the observed proteolytic cleavage, rather than simply time, temperature, or acidity, which can also cleave peptide bonds. Because this cleavage increases with alkalinity, the already known mechanism of peptide cleavage in dilute acids can be ruled out for this mechanism. To further delve into this phenomenon, we utilized mass spectrometry to analyze the specific cleavage sites. Identification of the TCEP Mediated Proteolysis Location by Mass Spectrometry As previously mentioned, the phenomenon of TCEP mediated proteolysis was first observed with SDS-PAGE analysis of recombinant L1. Mass spectrometry was employed to identify the proteolytic products. Recombinant L1 was incubated with 50 mM TCEP for 2 minutes at 80 o C, with 90 mM TCEP for 20 minutes at 80 o C, and, as a control which should reduce the disulfides without introducing cleavage, with 90 mM DTT for 60 minutes at 37 o C. The resulting chromatograms are shown in Supporting Information 35,697 35,695 315 D/C 50 mM TCEP, 2 min 90 mM TCEP, 20 min 39,152 39,150 348 D/Y 50 mM TCEP, 2 min 90 mM TCEP, 20 min 39,942 39.942 358 D/P, D/P 90 mM TCEP, 20 min 40,100 40,099 356 D/N 50 mM TCEP, 2 min 90 mM TCEP, 20 min 41,516 41,516 370 D/T 90 mM TCEP, 20 min 41,634 41,631 371 D/D 50 mM TCEP, 2 min 90 mM TCEP, 20 min 46,229 46,230 414 D/T 90 mM TCEP, 20 min 46,873 46,873 419 D/P 50 mM TCEP, 2 min 90 mM TCEP, 20 min 48,883 48,882 437 D/P 50 mM TCEP, 2 min 90 mM TCEP, 20 min 51,138 51,138 456 D/L 90 mM TCEP, 20 min 51,366 51,366 458 D/Q 90 mM TCEP, 20 min Table 1. Summary of cleavages observed cleavages and their assignments for the incubation of recombinant L1 with TCEP. Figure S1. The control shows no evidence of proteolytic degradation and the highest amount of intact L1 at approximately 3.5 minutes. Samples incubated with TCEP, on the other hand, show decreased levels of intact L1 and several lower intensity peaks ranging in retention times from 5 minutes to 7 minutes. Chromatograms were analyzed in 6-second intervals. The mass spectra within each interval were combined and the combined spectrum was subsequently deconvoluted. Resultant masses (Supporting Information Table S1 and Supporting Information Table S2) were compared to theoretical hydrolysis products of recombinant L1. As shown in Table 1, these experiments demonstrate that proteolytic cleavage occurs C-terminal to aspartic acid residues. Of the 11 identified fragments, six were observed with the 50 mM TCEP sample incubated for 2 minutes, while all 11 fragments were observed with the 90 mM TCEP sample incubated for 20 minutes. This observation further illustrates the aforementioned concentration and time dependence of this proteolytic pathway. Of particular interest is the 39,942 Da fragment. This product arises from clipping at two separate aspartic acid residues, generating a 358-residue long product. Interestingly, both aspartic acid residues are immediately proceeded by a proline residue. Yet, Table 1 reveals 7 other amino acids on the carboxyl side of aspartic acid, confuting any specificity towards the Asp-Pro moiety. While the exact mechanism of TCEP proteolysis is unknown, the observation of cleavages at a variety of Asp-X pairs within the recombinant L1 dataset suggests this pathway is prevalent for the C-terminal side of all aspartic acid residues, regardless of the proceeding amino acid. Additionally, the observance of this product, which arises from a double clipping event, suggests that incubation of proteins at high temperatures and high concentrations of TCEP may be used for complete digestion in bottom-up mass spectrometry workflows. Peptide Mapping of Protein Standards Peptide mapping experiments were performed with a set of seven standard proteins (lysozyme, BSA, myoglobin, carbonic anhydrase, alcohol dehydrogenase, phosphorylase b, and protein A) ranging in mass from approximately 14 kDa to approximately 98 kDa, with a total of 178 aspartic acid residues across all proteins. Here, samples were incubated overnight with 100 mM TCEP at 80 o C to assess sequence coverage and specificity of digestion. Cleavage C-terminal to 140 aspartic acid residues was observed. In this work, the efficiency of TCEP mediated hydrolysis is repre- Figure 5. Instances A) possible X-Asp and observed X-Asp cleavages, and B) possible Asp-X and observed Asp-X cleavages. sented as the percent of observed cleavages (140) compared to the possible cleavages (178) and was calculated to be 78.7%. Similar experiments were performed with trypsin using a conventional digestion protocol. Samples were reduced with DTT, alkylated with iodoacetamide, and digested overnight with trypsin at 37 o C. Cleavage at 231 of the possible 333 locations (Lys and Arg) was observed, resulting in an efficiency of 69.4%. For both TCEP and trypsin, overnight incubation resulted in a high degree of sequence coverage (Supporting Information Figure S2 through Supporting Information Figure S15), with greater than 54% sequence coverage observed for each of the standard proteins. In this analysis, only peptides with MS/MS data and zero missed cleavages were included. When expanding the parameters to include one missed cleavage and mass only peptides, the extent of sequence coverage increases (data not shown). Inherently, this would increase the efficiency for both TCEP and trypsin. Additionally, many of the missed cleavages are a result of the generation of small peptides which are often difficult to detect via LC-MS. Glutamic acid, like aspartic acid, is an amino acid with a carboxylic acid sidechain. While the HPV mass spectrometry data set suggests TCEP mediated hydrolysis occurs exclusively to the carboxyl terminus of aspartic acid residues, we sought to investigate whether this behavior also occurs at glutamic acid residues due to the highly analogous nature of the two. LC-MS data was reprocessed using identical parameters, this time searching for peptides generated via cleavage C-terminal to glutamic acid residues (Supporting Information Figure S16 through Supporting Information Figure 22). The glutamic acid dataset shows significantly lower coverage compared to the aspartic acid dataset. Additionally, most of the coverage arises from mass only peptide identifications. While TCEP mediated cleavage may also occur at glutamic acid, the low MS/MS sequence coverage suggests that this pathway is less favorable compared to its aspartic acid counterpart. In order to quantitatively investigate the propensity for TCEP induced cleavage at specific aspartic acid moieties, we analyzed the 140 cleavage events of the seven standard proteins. The number of possible cleavage events as well as the number of observed cleavage events is displayed in Figure 5. Data is deconstructed into specific X-Asp (preceding) and Asp-X (proceeding) pairs. When comparing the number of possible cleavages to the number of observed cleavages (compare light blue to dark blue), the Glu-Asp and Asp-Asn moieties appear to have a suppressive effect on TCEP mediated protein hydrolysis as evidenced by the largest differences in possible cleavage events to observed cleavage events. Figure 5A and Figure 5B illustrate a disparity in pair frequencies; that is to say, the specific pairs are not evenly distributed. Alanine, glutamic acid, and lysine are among the most prevalent preceding residues while methionine and tryptophan are the least prevalent. The identities of the proceeding residues are more evenly dispersed with the exception of cysteine, histidine, methionine, and tryptophan. To account for the differences in frequency, the propensity for each X-Asp and Asp-X pair was calculated by dividing the number of possible events by the number of observed events. As shown in Figure 6, the Glu-Asp, Met-Asp, Trp-Asp, Asp-Asn, and Asp-Ser pairs display low hydrolysis propensities. However, the methionine and tryptophan containing pairs only occur once throughout the dataset and there is not sufficient evidence to claim is these moieties have an inhibitory effect or is the missed cleavage is simply a result of the small sample size. Conclusions TCEP mediated protein hydrolysis has been demonstrated. This process occurs in a concentration, temperature, time, and pH dependent manner primarily on the carboxyl terminus of aspartic acid Figure 6. Propensity of TCEP mediated cleavage for A) X-Asp and B) Asp-X pairs. residues. The specificity of this pathway towards aspartic acid, and to a lesser extent, glutamic acid, suggests the carboxylic acid sidechain is involved in the hydrolysis reaction, though investigation of the specific mechanism was outside the scope of this work. It has been shown that incubation of common laboratory proteins with high concentrations of TCEP results in extensive sequence coverage and an efficiency of approximately 80%. Our data-driven analysis revealed that cleavage occurs at most aspartic acid residues regardless of the identity of the preceding and proceeding amino acids. Several missed cleavages were observed when the preceding amino acid was a glutamic acid residue, and more specifically at the Glu-Asp-Asn and Glu-Asp-Gly moieties. These moieties, however, were all located on Protein A in a region of low sequence coverage for both TCEP and trypsin incubated samples. Future experiments could incorporate site specific mutations to further investigate if these moieties are inhibitory to hydrolysis. Overall, this work demonstrates an alternative approach for peptide mapping workflows which eliminates the need for alkylation and buffer exchange and also serves as a cautionary tale to those utilizing TCEP at high concentrations and high temperatures. AUTHOR INFORMATION Corresponding Author Daniel S. McCracken – Vaccine Analytical Research & Development, Merck & Co., Inc., 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States; Phone: (215) 652-5419; E-mail: [email protected] Present Addresses Feras Hatahet: Protein Sciences, Vertex Pharmaceuticals, 3215 Merryfield Row, San Diego, CA 92121 Author Contributions ‡D.S.M. and D.J.F. contributed equally to this work. D.S.M. and D.J.F. collected data and engaged in the drafting and editing of the manuscript. K.G.E. contributed to the method development necessary for these experiments. F.H. engaged in data interpretation and edited the manuscript. All authors have given approval to the final version of the manuscript. Notes The authors declare the following competing financial interest: At the time experiments were conducted, all authors were employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. ACKNOWLEDGMENT The authors acknowledge Tiago Matos, Danielle Miller, and Benjamin Roose for their brainstorming, contributions to experimental design, and troubleshooting. The authors also acknowledge Alyssa Stiving for her assistance with peptide mapping and helpful discussion of mass spectrometry data. REFERENCES 1. Liu, P.; O’Mara, B. W.; Warrack, B. M.; Wu, W.; Huang, Y.; Zhang, Y.; Zhao, R.; Lin, M.; Ackerman, M. S.; Hocknell, P. K.; Chen, G.; Tao, L.; Rieble, S.; Wang, J.; Wang-Iverson, D. B.; Tymiak, A. A.; Grace, M. J.; Russell, R. J., A Tris (2-Carboxyethyl) Phosphine (TCEP) Related Cleavage on Cysteine-Containing Proteins. Journal of the American Society for Mass Spectrometry 2010, 21 (5), 837-844.2. Rhee, S. S.; Burke, D. 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R.; Mohammed, S., Lys-N and Trypsin Cover Complementary Parts of the Phosphoproteome in a Refined SCX-Based Approach. Analytical Chemistry 2009, 81 (11), 4493-4501.37. Mohammed, S.; Lorenzen, K.; Kerkhoven, R.; Breukelen, B. v.; Vannini, A.; Cramer, P.; Heck, A. J. R., Multiplexed Proteomics Mapping of Yeast RNA Polymerase II and III Allows Near-Complete Sequence Coverage and Reveals Several Novel Phosphorylation Sites. Analytical Chemistry 2008, 80 (10), 3584-3592.38. Ramachandran, L. K.; Witkop, B., [30] N-Bromosuccinimide cleavage of peptides. Methods in enzymology 1967, 11 , 283-299. Information & Authors Information Version history V1 Version 1 29 May 2025 Peer review timeline Published Rapid Communications in Mass Spectrometry Version of Record 14 Feb 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords hydrolysis mass spectrometry proteolysis redox tcep Authors Affiliations Daniel S. McCracken 0000-0001-8759-3539 [email protected] Merck & Co Inc West Point Research Library View all articles by this author David J. Foreman Merck & Co Inc West Point Research Library View all articles by this author Kyle G. Esposito Merck & Co Inc West Point Research Library View all articles by this author Feras Hatahet Merck & Co Inc West Point Research Library View all articles by this author Metrics & Citations Metrics Article Usage 731 views 265 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Daniel S. McCracken, David J. Foreman, Kyle G. Esposito, et al. TCEP-Mediated Protein Hydrolysis: Highly Selective Cleavage C-terminal to Aspartic Acid. Authorea . 29 May 2025. DOI: https://doi.org/10.22541/au.174849500.05592731/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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