Combination of SCX Fractionation and Charge-Reversal

Article Cite This: J. Proteome Res. 2019, 18, 2954−2964

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Combination of SCX Fractionation and Charge-Reversal Derivatization Facilitates the Identification of Nontryptic Peptides in C‑Terminomics Patrick Kaleja,† Andreas O. Helbig,† and Andreas Tholey*,† †

Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105 Kiel, Germany

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S Supporting Information *

ABSTRACT: The proteome wide, mass spectrometry based identification of protein C-termini is hampered by factors such as poor ionization efficiencies, low yielding labeling strategies, or the need for enrichment procedures. We present a bottom-up proteomics workflow to identify protein C-termini utilizing a combination of strong cation exchange chromatography, on-solid phase charge-reversal derivatization and LC−MS/MS analysis. Chargereversal improved both MS and MS/MS spectra quality of peptides carrying nonbasic C-terminal residues, allowing the identification of a high number of noncanonical C-termini not identified in nonderivatized samples. Further, we could show that C-terminal 18O labeling introduced during proteolytic processing of the samples is not suitable to distinguish internal from Cterminal peptides. The presented workflow enables the simultaneous identification of proteins by internal peptides and additionally provides data for the C- and N-terminome. Applying the developed workflow for the analysis of a Saccharomyces cerevisiae proteome allowed the identification of 734 protein C-termini in three independent biological replicates, and additional 789 candidate C-termini identified in two or one of three biological replicates, respectively. The developed analytical workflow allowed us to chart the nature of the yeast C-terminome in unprecedented depth and provides an alternative methodology to assess C-terminal proteolytic protein processing. KEYWORDS: cyanogen bromide, 18O-labeling, PICS, proteolysis, ragged peptides, solid-phase derivatization, TAILS, trypsin specificity, fragmentation, S. cerevisiae

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polymer.6 Other workflows are based on retention time shifts of internal peptides in chromatography, for example, by the charge-based separation of different peptide species in strong cation exchange (SCX) chromatography, which is exploited in C-terminal COFRADIC.7 Here, native C-termini are enriched by retention time shift, since they mostly show a reduced insolution charge state compared to trypsin-generated peptides, which carry a basic residue at the C-terminus. In parallel, this also enriches for other lower charged species, for example, Nacylated peptides.8 Further, compared to N-terminal approaches, there are only few enrichment strategies for Cterminal peptides after labeling (e.g., ProC-TEL9 ), or approaches mimicking N-terminal enrichment strategies for C-terminal peptides.10 In order to label and enhance peptide ionization, we recently introduced a charge reversal workflow, which is based on the introduction of a positive charge at the intact protein Cterminal carboxyl group via derivatization with N,N-dimethylethylenediamine (DMEDA).11

INTRODUCTION The identification of protein N- and C-termini in proteomes is major prerequisite for the elucidation of intra- and extracellular proteolytic processes. A number of approaches have been developed and successfully applied for the analysis of protein N-termini, for example, N-TAILS,1 COFRADIC 2 and ChaFRADIC.3 Generally, N-terminomics profits from numerous established approaches for the chemical modification of amino groups. In contrast, C-terminome analysis is hampered by some inherent drawbacks, for example problems encountered with the activation and derivatization of carboxyl groups, or the less efficient ionization of C-terminal peptides, for example, compared to internal peptides generated by tryptic digests. A related problem is the identification of nontryptic peptides in classical proteomics as peptides with nonbasic C-terminal residues are known to be less efficiently analyzed both MS and MS/MS due to the lack of a positive charge at the Cterminus.4,5 Several approaches to improve identification of C-terminal peptides have been developed and applied successfully. In the C-TAILS approach, C-terminal peptides are enriched by the depletion of internal peptides via covalent immobilization on a © 2019 American Chemical Society

Received: April 23, 2019 Published: June 13, 2019 2954

DOI: 10.1021/acs.jproteome.9b00264 J. Proteome Res. 2019, 18, 2954−2964

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Journal of Proteome Research

Figure 1. Overview of the workflow for of the proteome-wide identification of protein C-termini. Step 1: Digestion of proteins using trypsin in 18Owater leads to the incorporation of 18O at the tryptic peptides C-termini (internal peptides). Step 2: Fractionation of the digest by SCX according to in-solution charge states of +1 (N-acylated and C-terminal peptides), +2 and +3/+n. Step 3: Desalting followed by on-solid phase derivatization (amino group dimethylation and carboxyl group EDC/NHS/DMEDA derivatization) of half of the fractions from step 2; the other half is analyzed without derivatization. Step 4: After LC−MS analysis 18O incorporation of peptides is determined. 18

Aside from such chemical strategies, enzyme catalyzed labeling procedures are very attractive as they can be easily implemented in bottom up workflows and circumvent poor Cterminal chemical labeling and enrichment efficiencies. Trypsin catalyzed proteolysis of proteins in 18O-water leads to the incorporation of one or two 18O atoms at the peptide Cterminus.12 This leads to a mass shift of either +2 m/z or +4 m/z of internal peptides compared to the original protein Cterminus, which should remain unlabeled. This mass difference can easily be detected on MS1 and MS2 level allowing differentiation of enzymatically derived peptides and original C-termini and hence 18O labeling has been successfully applied for the identification of protein C-termini in numerous studies.13,14 However, to our knowledge, large scale use of

O labeling for full proteome/C-terminome analysis has not yet been reported. Despite significant advances achieved with the aforementioned C-terminomics methods, it is a noteworthy observation that only a relatively small number (compared to the expected number) of C-termini are commonly identified. For example, using the C-TAILS approach, Schilling et al. identified 460 Ctermini in an Escherichia coli proteome.6 A similar number of canonical C-termini were identified in the same bacterium by the use of charge reversal modification in combination with two protease digestions (424 C-termini),11 whereas an alternative multienzyme digestion encompassing five different proteases, was reported to identify 722 canonical C-termini in the E. coli proteome.15 These numbers demonstrate the need 2955


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Sep-Pak C18 cartridges from Waters (Eschborn, Germany) with acetonitrile and subsequently lyophilized.

for more efficient procedures to increase the coverage of Cterminomes. We here report a novel approach, which combines different elements of the aforementioned C-terminomics strategies. The workflow (Figure 1) is based on tryptic digestion of the whole proteome in 18O-water and charge-based separation using SCX, on-solid phase charge reversal derivatization of the +1, +2, and +3 charged SCX-fractions, prior to LC−MS analysis. Using the proteome of the baker’s yeast (S. cerevisiae), we investigated the suitability of the 18O labeling for unambiguous distinction between internal and C-terminal peptides in full proteome analysis. We demonstrate that the commonly used 18 O labeling strategy has severe drawbacks in large scale bottom-up proteomics based C-terminomics. Further, an indepth analysis of the MS/MS properties of charge-reversal derivatized peptides was performed enabling the identification of a high number of canonical and putative processed (truncated) C-termini.

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SCX Chromatography for Charge State Separation and DMEDA On-Solid Phase Derivatization

SCX chromatography was performed on a Dionex Ultimate 3000 (Thermo Scientific) with a 200 × 2.1 mm, 5 μm, 300 Å Polysulfethyl A SCX column by PolyLC Inc. (Columbia, MD). Samples were loaded in eluent A (5 mM KH2PO4, 30% ACN, pH 2.7 HCl). Peptides were eluted with a KCl gradient (eluent B: as A containing 350 mM KCl) and NaCl (eluent C: as A containing 800 mM NaCl) at a flow rate of 250 μL/min.3 Gradient: isocratic 0% B from 0 to 10 min, followed by a linear ramp to 20% B up to 40 min, 30% B up to 50 min, 50% B up to 55 min, 100% B at 60 min. At 69 min the system was switched to 100% C for 5 min. Peptide in-solution charge states were identified in pre-experiments and fractions collected as following: +1 (0 to 24 min), +2 (25 to 40 min), +3 (41 min to end) (Supporting Information (SI) Figure S1). Charge state fractions were dried, redissolved in 5% formic acid and loaded onto a Sep-Pak C18 cartridge. After washing with 3 mL 5% formic acid, the underivatized sample was eluted with acetonitrile. The samples to be derivatized were reductively dimethylated when bound to solid phase at the Sep-Pak cartridges.17 On-solid phase DMEDA derivatization was performed with 5 mL of freshly prepared 50 mM EDC, 10 mM NHS and 75 mM DMEDA in 500 mM MES buffer pH 5 over 60 min similar to Somasundaram et al. 2016. The column was rinsed with 5% formic acid and peptides eluted with acetonitrile. Finally, all sample groups were lyophilized to dryness.

EXPERIMENTAL PROCEDURES

Chemicals

Sequencing grade modified trypsin was purchased from Promega (Madison, WI) and 18O labeled water (labeling grade, 97%) by euriso-top (Saarbrücken, Germany). Pierce BCA protein assay kit was purchased from Thermo Scientific (Bremen, Germany). Deionized water (18.2 MΩ/cm) was prepared by an arium 611VF system (Sartorius, Göttingen, Germany). Yeast media were autoclaved or sterile filtered with pore sizes of 0.2 μm (Sartorius, Göttingen) prior of use. Other chemicals were purchased from Sigma-Aldrich (Munich, Germany). Yeast Cell Culture, Lysis, And Tryptic

Mass Spectrometry Analysis and Data Evaluation

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O Digest

Peptides were resolubilized in 0.05% formic acid and used for downstream reverse phase low pH nanoHPLC online coupled to an Orbitrap Q Exactive Plus Mass Spectrometer (Thermo Scientific). Prior to chromatographic separation, peptides were desalted on a C18 trapping μ-precolumn Acclaim PepMap 100 with 300 μm i.d. × 5 mm, 100 Å pore size and 5 μm particle size (Thermo Scientific). Nano-LC was performed on an Acclaim PepMap RSLC 75 μm × 50 cm nanoViper column (Thermo Scientific) with a Dionex Ultimate 3000 System (Thermo Scientific) and eluent A (0.05% formic acid in deionized water) and B (30% acetonitrile, 0.05% formic acid in deionized water). Gradient started at 5% eluent B up to 2 min, following a linear increase to 20% at 80 min, 30% at 110 min, 50% at 122 min and to 95% at 127 min. For online measurements, a data dependent acquisition method with top 15 precursors for higher energy collisional dissociation (HCD) fragmentation at 29% normalized collision energy in positive ion mode was utilized. For pools of the SCX+1 fractions, peptide fragmentation of charge states +1 to +8 was enabled, whereas SCX+2 and +3 was set to +2 to +8 ion charge. Resolution on MS1 was set to 70 000 with a scan range from 300 to 1800 m/z and on MS2 to 17 500. AGC target was set to +3 e6 on MS1 level and +1 e5 on MS2, respectively. Dynamic exclusion list was used with 40 s cycles and exclusion of isotopes set true. The isolation window was selected with 3 m/z. Data analysis was performed on Proteome Discoverer 2.2 (Thermo Scientific) with SequestHT search nodes and Cterminal precursor quantification methods without data normalization based on ion intensity. For nonderivatized samples 18O labeling (18O(0) [+0 Da], 18O(1) [+2.0042 Da]

Yeast cells (Saccharomyces cerevisiae, strain BY4742, Euroscarf) were cultivated as independent, biological replicates in synthetic yeast nitrogen base medium with amino acids media (0.67% nitrogen base medium, 2% glucose, 0.19% medium supplement in deionized water) to OD 3 at 600 nm.16 For short-term storage, cells were washed with PBS (Dulbecco’s Phosphate Buffered Saline, Sigma-Aldrich) and stored at −80 °C until use. For lysis, 300 mg (wet weight) of cells per replicate were transferred into 250 μL lysis buffer (6 M GndHCl, 100 mM HEPES pH 7.5, 2× cOmplete protease inhibitor) and lysed via a sonication probe (Bandelin Sonoplus; BANDELIN electronic GmbH & Co. KG, Berlin, Germany) in six cycles of 20 s with 100% power on ice. Cell debris was separated by centrifugation (4 °C for 20 min at 21 000g), washed with PBS, centrifuged and both supernatants pooled. Proteins were reduced via 40 mM DTT for 1 h at 56 °C and alkylated under light protection with 80 mM IAM for 1 h at room temperature. For protein precipitation, nine volumes of ethanol (−20 °C) were added to the sample and vigorously vortexed. The solution was stored for 16 h at −20 °C for precipitation. The precipitate was washed two times with ethanol and resolubilized by the addition of 100 μL 100 mM sodium hydroxide in H218O (18O water), which was subsequently diluted to 1 mL with 100 mM ammonium bicarbonate buffer pH 8 in 18O water. Protein quantification was performed via BCA assay using Pierce BCA assay kits from Thermo Scientific (Bremen, Germany). Protein digestion was performed with 4 mg sample per replicate utilizing trypsin (1:100 enzyme to protein ratio) at 37 °C for 16 h. Peptides were purified using 2956


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Journal of Proteome Research and 18O(2) [+4.0085 Da]) on peptide C-termini were set as variable modifications and quantification channels. Further variable modifications were oxidation on methionine [+15.9949 Da], acetylation on N-termini [+42.0106 Da] and as a fix modification carbamidomethylation on cysteine [+57.0215 Da]. For derivatized samples, DMEDA 18O labeling (DMEDA C-terminus [+70.0895 Da] and DMEDA 18O Cterminus [+72.0937 Da]) were used as fixed modifications and as quantification channels, respectively. Fixed modifications were set for dimethylation on lysine [+28.0313 Da] and carbamidomethylation on cysteine [+57.0215 Da], while variable modifications were set for acetylation on N-termini [+42.0106 Da], dimethylation on N-termini [+28.0313 Da], oxidation on methionine [+15.9949 Da] and DMEDA on aspartate and glutamate [+70.0895 Da]. Enzyme specificity was set to semitryptic with maximum two missed cleavages. Precursor mass tolerance was set to 10 ppm and fragment mass tolerance to 0.02 Da. Data were searched against a database consisting of 6721 yeast proteins (https://www.uniprot.org/ uniprot/; only reviewed sequences, 17th of May 2017). Derivatization efficiencies were calculated as previously described.11

Raw Data Repository

All LC−MS data have been deposited to the ProteomeXchange Consortium23 via the PRIDE partner repository with the data set identifier PXD013486. Instrument data were converted to prot.xml and pep.xml via Proteome Discoverer 2.2.

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RESULTS AND DISCUSSION

Charge Reversal Derivatization Enhances Identification of Nontryptic Peptides

After tryptic digestion of the S. cerevisiae proteome in heavy ( 18O)-water, the generated peptides were fractionated according to their in-solution charge states using SCXchromatography. This step follows two rationales: First, together with later LC−MS analysis, sample complexity is reduced. Second, by separation based on in-solution charges of the analytes, an enrichment of C-terminal peptides was intended. Due to the application of trypsin, all internal peptides, with the exception of N-acylated, are expected to elute in fractions corresponding to charges of +2 or higher. Vice versa, C-terminal peptides are expected to elute in the +1 charged fraction. However, three important deviations may occur for C-terminal peptides with (i) missed cleavages, (ii) histidine residues and (iii), such possessing a C-terminal lysine or arginine residue that might shift those to higher charged SCX fractions. SCX-fractions were split in half for two complementary strategies. One half was used for C18 cleanup and analyzed directly via online LC−ESI MS/MS (nonderivatized samples; SI Table S1). For the second half (derivatized samples), carboxyl groups were additionally derivatized with DMEDA, introducing a positively charged group at the peptide Cterminus (charge reversal derivatization; SI Table S2). By comparing the total ion chromatograms (TIC) of nonderivatized and derivatized samples, we observed clear changes in peptide elution profiles (SI Figure S2A−C). In derivatized samples of SCX+1 and SCX+2 fractions, peptides started eluting after 20 min, compared to 40 min for nonderivatized samples. As described earlier,11 C-terminal charge-reversal derivatization alters, and in most cases improves, peptide ionization behavior. Accordingly, strongest MS-signals were obtained for early eluting peptides in DMEDA modified samples. Interestingly, SCX+3 fractions showed the smallest retention time shifts and overall lower signal intensities for derivatized samples. We conclude that for higher (in-solution) charged peptides the effect of DMEDA derivatization seems to be less efficient or potentially negatively affecting peptide ionization. The effects of DMEDA derivatization on MS/MS results are described below. In the nonderivatized samples, taking into account both light and heavy (18O) C-termini, the number of identified peptides in SCX+1 was lower compared to SCX+2 and SCX+3 (2377 compared to 10 272 and 7717 peptides, respectively) (SI Table S3). Interestingly, 83.5% of the peptides identified in the SCX +1 fraction carried a C-terminal lysine or arginine residue (1985 peptides, vs 392 with nonbasic C-terminal residue). This is an only slightly reduced percentage compared to SCX+2 (95.3%) and +3 fractions (94.5%). Among the peptides identified with C-terminal Lys/Arg residues in fraction SCX+1, 13.3% were N-acetylated, which explains their occurrence in this fraction (SI Table S4).

Data Analysis and Visualization

For identification of C-terminal peptides in nonderivatized and DMEDA derivatized samples, three biological replicates were analyzed by bottom-up LC−MS. False discovery rates for PSM and peptide groups were calculated based on a reversed decoy database and validated via Percolator node with target FDR of 0.01 as a high confidence peptide group filter.18 For differentiation of protein C-termini and tryptic/internal peptides, a multifactor filter was used. 18O incorporation was determined by algorithm assigned peptide group modifications, as well as manually verified for multiple spectra. Combined results of replicates and SCX fractions were also tested and used as filter instrument. Venn diagrams were generated with help of BioVenn19 and Heatmaps using iceLogo.20 To analyze b- and y- ion series formation, annotated PSMs of nonderivatized and derivatized conditions were extracted as peak lists in text format and processed via an in house python script. The script matched PSMs to peptides and summarized identified b- and y-ions for each peptide. Boxplots for truncated peptides and tryptic-like C-termini were calculated and visualized, as well as statistical Wilcoxon-signed rank test performed. Determination of Trypsin Specificity by PICS

For trypsin cleavage site analysis using the PICS approach,21 yeast lysates were reduced and alkylated, followed by chemical digestion using cyanogen bromide at a chemical to protein weight ratio of 100:1 at room temperature for 18 h. Resulting peptides were on column desalted, dimethylated and dried. Subsequently, peptides were digested using trypsin in identical conditions as the main workflow. Tryptic activity was stopped by heat inactivation22 and neo N-terminal peptides removed by first labeling with NHS-disulfide biotin reagent, followed by incubation with Neutravidin. Peptide flow-through was collected and split for two analytical strategies: (I) one-half was desalted and analyzed without further processing (nonderivatized sample); (II) the other half was desalted, followed by on column EDC/NHS/DMEDA derivatization. Resulting peptides were conducted to LC−MS/MS analysis as previously described. Data analysis of both conditions are described in SI. 2957


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Figure 2. Changes in identified b- and y-ions for nonderivatized and DMEDA derivatized peptides. A: Box plots of the number of identified b- and y-ions for peptides identified in both conditions (nonderivatized and derivatized) calculated from annotated PSMs. Data were median summarized at peptide level and visualized as boxplots. For peptides without C-terminal lysine or arginine, a significant increase in y-ion formation can be seen after derivatization (median: 11 y-ions compared to 18.5, p = 3.1 × 10−39) and a decrease in b-ions (median: 17 compared to 14, p = 5.0 × 10−16). Compared to peptides with C-terminal lysine or arginine, no clear difference can be seen after derivatization (3887 peptides with K/R at Cterminus, 361 peptides without C-terminal K/R; nonderivatized: 72,002 PSMs; derivatized: 62,038 PSMs; Error bars indicating standard deviation, Wilcoxon-signed rank test). B−C: Annotated spectra of the peptide EILSGFHNAGPVAG in (B) nonderivatized and (C) derivatized samples.

and +3; SI Figure S2E,F). This can be attributed to the fact, that peptides with basic C-termini are effectively enriched in these SCX-fractions. In contrast, the charge-reversal derivatization significantly enhances the detection of peptides carrying nonbasic C-termini, which is, with the exception of proteins displaying C-terminal Lys/Arg residues, typical for protein Cterminal peptides. In contrast to our earlier study,11 which employed chargereversal derivatization at intact protein level, in the present approach the derivatization was performed at peptide level. The applied on-solid phase workflow is fast (within 1 h) and allows to perform other necessary steps, for example, reductive dimethylation of amino groups, with easy removal of excess reagents. Derivatization efficiencies for the C-terminus were at comparable high 84.6%, while efficiencies for derivatization on side chains of Asp/Glu were at a lower 52.5%. We did not observe significant side reactions, for example, esterification of hydroxyl-amino acids, during NHS/EDC-mediated chargereversal derivatization. Further, the high yields of reductive dimethylation, which can be regarded as almost quantitative in our protocol, also prevented the reaction of DMEDA with (free) amino groups.

We identified a low number of PSM in SCX+1 fractions deriving from singly charged precursor ions. Here, 2% of all PSM in SCX+1 fractions were solely identified by precursors with gas phase charge states of +1, while the majority of peptides was also present in higher charge states. In the DMEDA derivatized SCX+1 fraction, however, only 30.9% displayed a C-terminal Lys/Arg residue (617 peptides), indicating that charge reversal derivatization indeed facilitates mass spectrometric detection of nontryptic peptides (1383 peptides). In contrast, peptides with C-terminal Lys/Arg residues in SCX+2 and SCX+3 were present at similar levels to underivatized samples (91.4% and 94.5%). Numbers of identified peptides in the derivatized SCX+1 fraction were still lower compared to SCX+2 but higher than in SCX+3 (2000 compared to 3370 and 1746 peptides, respectively) (SI Table S3). Comparing peptide identifications in SCX+1 fractions of nonderivatized and derivatized samples, we found only a small overlap of 668 peptides, whereas 1709 peptides were solely identified in nonderivatized and 1332 peptides only identified in derivatized samples (SI Figure S2D). Thus, nonderivatized and derivatized samples delivered complementary information in SCX+1 fraction. In the SCX+2 and SCX+3 fractions, significantly higher numbers of peptides were identified in nonderivatized samples and most of the peptides identified in derivatized samples overlapped with those of nonderivatized samples. In consequence, charge-reversal derivatization led only to a relatively low number of additional identifications in the higher charged fractions (SI Figure S2E,F). In summary, peptide overlap and gain of DMEDA modification is most beneficial for nontryptic peptides in the SCX+1 fractions, but reduced for higher SCX factions (SCX+2

Charge-Reversal Derivatization Leads to Increased y-Ion Formation During HCD and Higher Peptide Coverage by Fragment Ions

The introduction of a positively charged group at the Cterminal carboxyl group (charge-reversal) has been shown to enhance MS spectra intensities of C-terminal peptides,11 with the asymmetric diamine N,N-dimethyl-ethylenediamine (DMEDA) delivering best results among different reagents tested. While in our previous study the effects on the MS/MS 2958


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of our study, three major critical issues were identified hampering this strategy: high variations in 18O incorporation levels, oxygen in-exchange and back exchange. First, we analyzed the 18O incorporation in nonderivatized samples, including both tryptic internal and protein C-terminal peptides. A high portion of peptides were identified in multiple forms with different numbers of heavy oxygen incorporated (18O(0), 18O(1), 18O(2)) (SI Figure S8). The majority of peptides were either identified solely with single 18O incorporation (18O(1): 6217 peptides, 35.2%), no incorporation (18O(0): 3346 peptides, 18.9%) or both labels (18O(0) and 18O(1): 4428 peptides, 25.1%). Only 507 peptides (2.9%) were identified as a single form with two oxygen atoms incorporated; 3178 peptides (17.9%) had two heavy oxygen atoms incorporated but were also identified as species with other 18O incorporation levels. In order to rule out potential problems with labeling, we performed control experiments with different batches of heavy water, all leading to similar incorporation patterns. Second, software based spectra interpretation identified 54 canonical C-terminal peptides with 18O incorporation, of which 34 (63.0%) displayed a Lys or Arg as C-terminal amino acid. Manual inspection of the spectra confirmed 18O incorporation as shown for example in the peptide NVWSYSR from the 40S ribosomal protein S21−B/(Q3E754) (SI Figure S9). For peptides with C-terminal Lys/Arg residues a binding to the active site of the serine protease can be a potential reason for the incorporation of a heavy oxygen atom, and thus lead to mis-annotation of C-terminal peptides as internal peptides. As a third critical issue we identified 18O back exchange, even when we applied conditions known widely to minimize this effect.22,24,25 We identified numerous − supposedly − internal peptides (with C-terminal Lys/Arg residues) which showed no or only incomplete 18O incorporation. These peptides can be either real tryptic peptides from which, by back-exchange, the isotope label has been lost, or they can be truncated (noncanonical) versions of proteins present in the biological sample. These two situations cannot be distinguished at peptide level unless back-exchange would be completely absent, which it is not. The 18O incorporation also has a direct influence of the DMEDA derivatization strategy. Since this derivatization replaces one oxygen from the C-terminus, the heavy isotope labeling can be completely lost. This directly renders 18Olabeling incompatible with the DMEDA-charge reversal derivatization strategy. Overall, we conclude that 18O labeling is not suitable in large scale C-terminomics approaches, both without or with Cterminal charge-reversal derivatization. Our results are in concordance with previous studies which show that trypsin catalyzed 18O incorporation generates diverse isotope signals, depending on peptide characteristics.26 Further, complete 18O labeling (18O(2)), either requires long incubation times or decoupled labeling steps,27,28 which is counterproductive for our analysis as this would also label the original C-termini, for example, via the above stated in-exchange. However, in selected cases, 18O labeling may be a useful additional parameter for validation of C-terminal identification, that is, for canonical peptides with nonbasic C-terminal residues.

spectra have been studied on a number of model peptides, here we analyzed hundreds of peptides in a proteome wide analysis, including both internal and C-terminal peptides. Comparing the MS/MS spectra of nonderivatized and DMEDA-derivatized C-terminal peptides, we observed overall changes in the number of identified y-ions for derivatized peptides compared to their nonderivatized counterparts. Two examples are shown in Figure 2 (with further examples shown in SI Figure S3−S6). While most nonderivatized peptides were identified with a prominent b-ion series, C-terminal DMEDA improved the coverage of y-ion series formation in HCD. For example, the nonderivatized peptide EILSGFHNAGPVAG (Figure 2B,C) was identified with each eight b- and y-ions, respectively, while the derivatized peptide was identified with 15 b- and 19 y-ions. We analyzed median summarized PSM data of all peptides, which were identified in both conditions (nonderivatized and derivatized) of our data set using an in-house python script: 3887 peptides with and 361 peptides without a basic Cterminal amino acid. The data revealed that derivatization of peptides without C-terminal Lys/Arg residues significantly increases the median number of identified y-ions from 11 to 18.5 ions after derivatization (p = 3.1 × 10−39, Wilcoxon-signed rank test). In contrast, b-ions become moderately less abundant (17 compared to 14 ions, p = 5.0 × 10−16; Figure 2A). Overall, this leads to the detection of significantly more fragment ions per peptide. Noteworthy, for C-termini with a terminal lysine or arginine, this effect after derivatization was significantly reduced (Figure 2A). Independent of the nature of the C-terminal amino acid, we observed a general increase in higher charged y-ions after derivatization, which was even stronger for higher charged SCX fractions (SI Figure S7). We assume this to be an effect of the DMEDA group, which adds to the basicity of the peptides and might increase y-ions charge states especially in higher charged SCX fractions. Overall, we conclude that DMEDA derivatization can improve fragmentation of peptides without C-terminal Lys/ Arg by leading to b- and y-ion distributions that resemble fragmentation patterns observed for tryptic peptides; we assume that this contributes to increased identification rates. Our data show that this potential becomes utilizable after a sample decomplexing in form of charge dependent strategies like SCX, which is vital to maximize the benefits of this methodology for nontryptic peptides. Trypsin Catalyzed C-Terminomics

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O-Labeling Is Not Suitable for

An inherent problem in C-terminomics studies is the evaluation of false positive/false negative assignments, as usually only the genetically encoded C-termini can be clearly assigned as correct identification. In contrast, truncated Ctermini, especially such which carry the same C-terminal amino acid as the recognition sequences of the protease used in the study (e.g., Lys/Arg for trypsin) are more difficult to confirm. Here, the incorporation of heavy oxygen during proteolysis could − theoretically − allow for a clear distinction between native (also truncated) C-termini and such formed during sample processing. While this strategy has been extensively employed for the analysis of single proteins in the past,13,14 to our knowledge, the present study is the first study performing this type of labeling in large scale LC−MS C-terminomics approaches on a proteome wide level. However, in the course 2959


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Journal of Proteome Research Combination of SCX and DMEDA-Charge Reversal Derivatization Increases the Number of Identified C-Termini

As 18O-labeling was shown not to be suitable for Cterminomics, we interpreted the data set taking into account both 18O-labeled as well as nonlabeled peptides; in cases a peptide was identified in both labeling states, it was counted as a single hit. A putative C-terminus was counted only once, even when it was covered by several peptides (e.g., missed cleavage sites or methionine oxidation) (SI Figure S10). In the following, we state the numbers of termini identified in three biological replicates, followed by the identifications in two and one replicates in brackets (2 replicates | 1 replicate). By utilizing these settings, we were able to identify 51 (20| 19) canonical C-termini in nonderivatized samples of SCX+1 fractions. After DMEDA derivatization these numbers were found elevated, with 108 (20|18) termini (Figure 3A,B, SI Figure 4. Canonical and noncanonical C-termini identified in nonderivatized and DMEDA derivatized samples in SCX+1 fractions. Shown are all termini identified in three of three biological replicates (upper row) or independent of the number of replicates (lower row). Termini are defined by every identified peptide group displaying the same C-terminal cleavage site in a known protein sequence independent of N-terminal prolonged sequences and/or modifications.

termini, with 49 solely identified in nonderivatized and 105 in derivatized samples (Figure 4). Interestingly, SCX+2 and SCX+3 fractions revealed further 43 in three replicates (27 in two|18 in one) and 43 (29|17) canonical C-termini in nonderivatized samples, and only 17 (6| 9) and 10 (5|14) C-termini after DMEDA derivatization, respectively. This displays again the inverse effect of charge reversal in higher charged SCX fractions. For the identification of C-termini being not equivalent with the genetically encoded ones (noncanonical termini), we excluded peptides with C-terminal Lys or Arg residues as these peptides cannot be distinguished from internal peptides generated by tryptic digestion in our workflow. Consequently, in a first step all peptides identified without C-terminal Lys/ Arg were assumed as putative C-terminal peptides. Second, we focused on SCX+1 fractions as these were the most expected ones to include C-termini. In nonderivatized samples, 101 putative noncanonical Ctermini fulfilling these criteria were identified in three biological replicates; additional 74 and 113 peptides were identified in two or one replicate, respectively Figure 3C, SI Figure S6). In DMEDA derivatized samples, 552 (311|356) putative noncanonical C-termini were identified (Figure 3C, SI Table S6). Taken these replicates together, 109 termini were solely identified without derivatization and 1040 solely after DMEDA derivatization (Figure 4). Most strikingly here is the 10-fold higher number of putative C-terminal peptides identified after DMEDA derivatization. Even considering only highest confidence peptides identified in three of three replicates, provided 600 C-termini across both condition. The SCX+2 and SCX+3 fractions revealed 116 (127|162) and 105 (99|116) C-termini in nonderivatized samples, while after charge-reversal derivatization 94 (84|85) and 13 (30|28) putative truncated C-termini could be identified. Thus, as

Figure 3. Number of biological replicates in which C-termini were identified in nonderivatized or derivatized samples. Termini are defined by every identified peptide group displaying the same Cterminal cleavage site in a known protein sequence independent of Nterminal prolonged sequences and/or modifications. The set presents canonical C-termini identified in SCX+1 fractions of (A) nonderivatized and (B) DMEDA derivatized samples; and noncanonical C-termini without C-terminal Lys/Arg present in (C) nonderivatized and (D) DMEDA derivatized samples.

Table S5). Combining both data sets, 134 canonical C-termini were identified in three replicates of SCX+1 fractions, with 25 termini overlapping between both analytical strategies, showing the complementarity of the approaches (Figure 4). Using additional identifications made in two and one biological replicates, respectively, revealed an overlap of 41 canonical C2960


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identified with up to five different C-termini. Using a short filter of two amino acids between every truncation event, we identified 49 proteins with at least one ragging event. The longest event being identified was in the 60S acidic ribosomal protein P2-beta ranging from position 70 to 83 encompassing 14 C-termini: LAAVPAAGPA| 1 S| 2 A| 3 G| 4 G| 5 A| 6 A| 7 A| 8 A| 9 S|10G|11D|12A|13A|14.

described above, benefits of charge-reversal derivatization again diminished at increasing in-solution charge states. A closer inspection of the C-terminal amino acid residues of the identified C-termini in DMEDA derivatized samples revealed a high number of peptides with hydrophobic residues (e.g., Ala, Phe, Tyr, Met, Leu; Figure 5) relative to the natural amino acid frequencies in the S. cerevisiae proteome.

Charge Reversal Derivatization Increases Peptide Identifications with Hydrophobic Amino Acids

The increased frequency of the occurrence of hydrophobic Cterminal residues, especially Phe, Tyr, Leu, Met, in the putative C-terminal peptides could potentially hint for side activities in the work protease employed. Chymotryptic and pseudotryptic activities are known side activities observed in trypsin catalyzed reactions, mainly caused by impurities in the enzyme preparations or by increased reaction times. Chymotrypsin hydrolyzes preferably peptide bonds C-terminal to aromatic or hydrophobic residues (Phe, Tyr, Trp, Leu, Met). For pseudotrypsin, the major cleavage occurs C-terminal to lysine and arginine residues, but also to a minor extend C-terminal to Leu/Phe/Tyr/Met.29 However, we used a protease which was TPCK treated, which blocks any residual chymotryptic activity. Further, the protease was reductively methylated, which significantly reduces autoproteolysis of trypsin, which is the major source of pseudotrypsin formation.29 In order to test this hypothesis, we performed a “proteomic identification of protease cleavage sites” (PICS) experiment,21 which is a method to identify preferred residues for cleavage catalyzed by the utilized protease. We employed a peptide library built by cyanogen bromide digestion of a yeast proteome, and performed a parallel analysis of nonderivatized and DMEDA-derivatized peptides generated by the target protease (SI Tables S7, S8). As shown in Figure 5, the major cleavage site identified was C-terminal to Arg, whereas cleavage at the reductively dimethylated Lys residues was only very weak. Most importantly, only a very low number of peptides with other C-terminal amino acids was identified, both in the nonderivatized and the DMEDA-PICS samples. This reflects that the trypsin used does not exhibit significant side activities at the same reaction conditions used in our main strategy (Figure 1). Hence, we conclude that the majority of 600 noncanonical C-termini identified with the SCX/nonderivatized/DMEDA derivatized approach can be regarded as true positive identifications of − at least putative − truncated C-termini. In our previous study we identified 157 noncanonical Ctermini in E. coli,11 which is in the same range as reported by Zhang et al. (176)15 and Schilling et al. (113)6 for the same organism. Thus, the DMEDA derivatization on peptide level seems to be capable of identifying a larger number of truncated C-termini. Interestingly, the number of putative truncated Ctermini comes close to such described in N-terminomics studies, which report about 50% of their termini to be noncanonical.30 However, even taking into account that our filters neglect further candidates (e.g., C-terminal Lys/Arg-peptides), still the number of identified C-termini remains below that of expected termini when one takes into account the number of identified proteins in these organisms in classical proteomics studies.

Figure 5. Heatmap of C-terminal amino acid frequencies in analyzed data sets relative to natural abundance in S. cerevisiae proteome at significant values of p = 0.01. Displayed are noncanonical C-termini identified in 3/3 replicates within nonderivatized and DMEDA derivatized samples, as well as results of PICS analysis in nonderivatized and DMEDA derivatized conditions. Termini are defined by every identified peptide group displaying the same Cterminal cleavage site in a known protein sequence independent of Nterminal prolonged sequences and/or modifications.

Analyzing our data set revealed the 600 putative noncanonical C-termini to be linked to only 245 gene products, implying multiple C-termini for single proteins. While for 151 proteins one noncanonical terminus was identified, 94 proteins were identified with a total of 449 termini. For a number of proteins, these termini resembled truncation events within short proximity range of single amino acids, which was also described in earlier C-terminomics studies.6,15 For example, Zhang et al. reported single proteins in their data set to be 2961


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Journal of Proteome Research Peptide Level Derivatization Reveals Previously Unidentifiable C-Terminal Peptides while Still Providing Common Proteomics Depth Analysis

higher charged SCX-fractions, which goes, however, on cost of the overall measurement time. A further aspect to be discussed is the high proportion of putative noncanonical C-termini compared to the canonical ones identified in our study (SCX+1 fraction and 3/3 biological replicates: 134 canonical compared to 600 putative noncanonical C-termini) which has to be critically evaluated. As we cannot completely rule out some degree of foreign proteolytic activity of the protease trypsin under the conditions applied here, we regard the identification of these nontryptic, noncanonical peptides as true identifications. Nevertheless, we would classify these peptides as putative truncated/noncanonical candidates which have to be confirmed by alterative biochemical or proteomics methods. For instance, the direct analysis of intact proteoforms, without any proteolytic processing (top-down proteomics) would circumvent a number of uncertainties involved inherently in any bottomup approach.31 A critical factor in N-/C-terminomics analysis is the requirement of relatively high amounts of sample, which is typically in the range of 0.5−1 mg total protein. Improved approaches such as hydrophobic tagging assisted N-termini enrichment (HYTANE) allow N-terminomics experiments down to 10 μg total protein.32 We will elucidate the potential to reduce the sample amounts needed for our C-terminomics method in future experiments, for example, by using miniaturized SCX-chromatography. Overall, our approach presented here adds a valuable piece to the toolbox of proteomics and in particular for the analysis of truncated proteins which is a prerequisite to characterize the dynamic nature of the proteome in the context of functional genomics.

As most other C-terminomics strategies remove internal peptides via depletion, protein identifications are only based on the isolated C-terminal peptides. In contrast, our approach still enables typical bottom-up analysis covering the full proteome. As such, nonderivatized condition provided 2078 and DMEDA derivatization 836 protein groups (identifications based on at least 1 unique peptide and minimum 2 peptides). Further, we identified 631 N-termini across all replicates, with 544 peptides identified in nonderivatized samples, and 258 after derivatization. Of the later, 87 termini were solely identified after DMEDA derivatization (SI Table S9). These numbers encompass both canonical N-termini and N-terminal acetylated peptides. The majority of N-termini identified in the SCX+1 fraction were N-acetylated (99.3% in nonderivatized and 98.4% in DMEDA derivatized samples). Further, we identified also a high number of N-terminal peptides in SCX+2 (175 peptides in nonderivatized and 62 peptides in DMEDA derivatized) and SCX+3 (159 peptides in nonderivatized and 24 peptides in DMEDA derivatized).

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CONCLUSIONS The analytics of C-terminal, and more generally of nontryptic peptides, is disfavored compared to classical internal tryptic peptides in bottom-up proteomics. Therefore, we setup a bottom-up workflow encompassing a charged based SCXfractionation and analyzed the peptides in parallel with or without charge reversal derivatization at the carboxy-terminus. The use of 18O labeling, introduced by proteolysis during sample processing, could be clearly shown to be not suitable for the distinction of internal from C-terminal peptides; factors such as in- and back-exchange prevented its use. In consequence, the unambiguous identification and assignment of presumably noncanonical C-termini is severely hampered. For this purpose, we suggest a two step filter to classify peptides as putative truncated (or noncanonical) C-termini: (i) the absence of the basic residues Lys or Arg at the C-terminus, as these residues are also recognized as cleavage sites for the work protease utilized; (ii) the occurrence of the peptides in the SCX+1 fraction. The exclusion of (putative) C-terminal peptides with Cterminal Lys/Arg residues represents a clear drawback of our strategy as a number of protease are known to cleave after basic residues; such truncation events will be clearly missed by our approach. The use of alternative proteases in sample processing could circumvent this problems, however, is not compatible with the overall strategy based on the prefractionation via SCX chromatography. The peptide focused charge-reversal derivatization strategy revealed high efficiencies and increased intensities, both in MS and MS/MS spectra, which facilitated the identification of peptides with nonbasic C-terminal amino acids. Using this approach, peptides not identified in the nonderivatized aliquots of the same became detectable. As both approaches deliver partially complementary results, the parallel analysis of nonderivatized and charge-reversal derivatized samples can be recommended. Further, DMEDA derivatization turned out to provide most benefits in the SCX+1 fraction, thus enhancing the MS based identification of peptides with low in-solution charges. Besides, there is additional gain of identifications in

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.9b00264. Data analysis for protease cleavage site identification (PICS). Figure S1: Elution profiles of SXC-chromatography. Figure S2: Total ion chromatograms and overlap in identified peptides of nonderivatized and DMEDA derivatized samples. Figure S3: MS/MS-spectra of the peptide LAAAQQQAQASGIMPSNEDVA with/without DMEDA. Figure S4: MS/MS spectra of the peptide LIDLTQFPAFVTPM with/without DMEDA. Figure S5: MS/MS spectra of the peptide IVSTEGNVPQTLAPVPYETFI with/without DMEDA. Figure S6: MS/ MS spectra of the peptide ILEDLVFPTEIVGK with/ without DMEDA. Figure S7: Box plots of the number of identified b- and y-ions for peptides identified in nonderivatized/derivatized peptides calculated from annotated PSMs. Figure S8: Nonderivatized peptides identified with different 18O-incorporations. Figure S9: MS spectrum demonstrating 18O in-exchange at peptides with C-terminal Lys/Arg. Figure S10: Visualization of the definition of C-terminal peptide identifications used in this study (PDF) Tables S1−S9: Summary on MS data and proteins/ peptides/C-termini identified (XLSX) 2962


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Nanoelectrospray Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Anal. Chem. 2000, 72 (6), 1179−1185. (14) Julka, S.; Dielman, D.; Young, S. A. Detection of C-Terminal Peptide of Proteins Using Isotope Coding Strategies. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2008, 874 (1), 101−110. (15) Zhang, Y.; Li, Q.; Huang, J.; Wu, Z.; Huang, J.; Huang, L.; Li, Y.; Ye, J.; Zhang, X. An Approach to Incorporate Multi-Enzyme Digestion into C-TAILS for C-Terminomics Studies. Proteomics 2018, 18 (1), 1700034. (16) Tucher, J.; Linke, D.; Koudelka, T.; Cassidy, L.; Tredup, C.; Wichert, R.; Pietrzik, C.; Becker-Pauly, C.; Tholey, A. LC−MS Based Cleavage Site Profiling of the Proteases ADAM10 and ADAM17 Using Proteome-Derived Peptide Libraries. J. Proteome Res. 2014, 13 (4), 2205−2214. (17) Boersema, P. J.; Raijmakers, R.; Lemeer, S.; Mohammed, S.; Heck, A. J. R. Multiplex Peptide Stable Isotope Dimethyl Labeling for Quantitative Proteomics. Nat. Protoc. 2009, 4 (4), 484−494. (18) Käll, L.; Canterbury, J. D.; Weston, J.; Noble, W. S.; MacCoss, M. J. Semi-Supervised Learning for Peptide Identification from Shotgun Proteomics Datasets. Nat. Methods 2007, 4 (11), 923−925. (19) Hulsen, T.; de Vlieg, J.; Alkema, W. BioVenn − a Web Application for the Comparison and Visualization of Biological Lists Using Area-Proportional Venn Diagrams. BMC Genomics 2008, 9 (1), 488. (20) Colaert, N.; Helsens, K.; Martens, L.; Vandekerckhove, J.; Gevaert, K. Improved Visualization of Protein Consensus Sequences by IceLogo. Nat. Methods 2009, 6 (11), 786−787. (21) Schilling, O.; Overall, C. M. Proteome-Derived, DatabaseSearchable Peptide Libraries for Identifying Protease Cleavage Sites. Nat. Biotechnol. 2008, 26 (6), 685−694. (22) Petritis, B. O.; Qian, W.-J.; Camp, D. G.; Smith, R. D. A Simple Procedure for Effective Quenching of Trypsin Activity and Prevention of 18O-Labeling Back-Exchange. J. Proteome Res. 2009, 8 (5), 2157− 2163. (23) Vizcaíno, J. A.; Deutsch, E. W.; Wang, R.; Csordas, A.; Reisinger, F.; Ríos, D.; Dianes, J. A.; Sun, Z.; Farrah, T.; Bandeira, N.; et al. ProteomeXchange Provides Globally Coordinated Proteomics Data Submission and Dissemination. Nat. Biotechnol. 2014, 32, 223− 226. (24) Niles, R.; Witkowska, H. E.; Allen, S.; Hall, S. C.; Fisher, S. J.; Hardt, M. Acid-Catalyzed Oxygen-18 Labeling of Peptides for Proteomics Applications. Anal. Chem. 2009, 81 (7), 2804−2809. (25) Stewart, I. I.; Thomson, T.; Figeys, D. 18O Labeling: A Tool for Proteomics. Rapid Commun. Mass Spectrom. 2001, 15 (24), 2456− 2465. (26) Ramos-Fernández, A.; López-Ferrer, D.; Vázquez, J. Improved Method for Differential Expression Proteomics Using TrypsinCatalyzed 18O Labeling with a Correction for Labeling Efficiency. Mol. Cell. Proteomics 2007, 6 (7), 1274−1286. (27) López-Ferrer, D.; Ramos-Fernández, A.; Martínez-Bartolomé, S.; García-Ruiz, P.; Vázquez, J. Quantitative Proteomics Using 16O/ 18O Labeling and Linear Ion Trap Mass Spectrometry. Proteomics 2006, 6 (S1), S4−S11. (28) Bantscheff, M.; Dü mpelfeld, B.; Kuster, B. Femtomol Sensitivity Post-Digest 18O Labeling for Relative Quantification of Differential Protein Complex Composition. Rapid Commun. Mass Spectrom. 2004, 18 (8), 869−876. (29) Keil-Dlouhá, V.; Zylber, N.; Imhoff, J.-M.; Tong, N.-T.; Keil, B. Proteolytic Activity of Pseudotrypsin. FEBS Lett. 1971, 16 (4), 291− 295. (30) Fortelny, N.; Pavlidis, P.; Overall, C. M. The Path of No ReturnTruncated Protein N-Termini and Current Ignorance of Their Genesis. Proteomics 2015, 15 (14), 2547−2552. (31) Tholey, A.; Becker, A. Top-down Proteomics for the Analysis of Proteolytic Events - Methods, Applications and Perspectives. Biochim. Biophys. Acta Mol. Cell Res. 2017, 1864 (11, Part B), 2191−2199. (32) Chen, L.; Shan, Y.; Weng, Y.; Sui, Z.; Zhang, X.; Liang, Z.; Zhang, L.; Zhang, Y. Hydrophobic Tagging-Assisted N-Termini

AUTHOR INFORMATION

Corresponding Author

*Phone: #49 (431) 500 30300; fax: #49 (431) 500 30308; email: [email protected]. ORCID

Andreas Tholey: 0000-0002-8687-6817 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS P.K. was supported by the Deutsche Forschungsgemeinschaft (DFG), project TH 872/9-1, A.H. by the SFB877, project Z2.

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REFERENCES

(1) Kleifeld, O.; Doucet, A.; auf dem Keller, U.; Prudova, A.; Schilling, O.; Kainthan, R. K.; Starr, A. E.; Foster, L. J.; Kizhakkedathu, J. N.; Overall, C. M. Isotopic Labeling of Terminal Amines in Complex Samples Identifies Protein N-Termini and Protease Cleavage Products. Nat. Biotechnol. 2010, 28 (3), 281−288. (2) Gevaert, K.; Goethals, M.; Martens, L.; Van Damme, J.; Staes, A.; Thomas, G. R.; Vandekerckhove, J. Exploring Proteomes and Analyzing Protein Processing by Mass Spectrometric Identification of Sorted N-Terminal Peptides. Nat. Biotechnol. 2003, 21 (5), 566−569. (3) Venne, A. S.; Vögtle, F.-N.; Meisinger, C.; Sickmann, A.; Zahedi, R. P. Novel Highly Sensitive, Specific, and Straightforward Strategy for Comprehensive N-Terminal Proteomics Reveals Unknown Substrates of the Mitochondrial Peptidase Icp55. J. Proteome Res. 2013, 12 (9), 3823−3830. (4) Tabb, D. L.; Smith, L. L.; Breci, L. A.; Wysocki, V. H.; Lin, D.; Yates, J. R. Statistical Characterization of Ion Trap Tandem Mass Spectra from Doubly Charged Tryptic Peptides. Anal. Chem. 2003, 75 (5), 1155−1163. (5) Burkhart, J. M.; Schumbrutzki, C.; Wortelkamp, S.; Sickmann, A.; Zahedi, R. P. Systematic and Quantitative Comparison of Digest Efficiency and Specificity Reveals the Impact of Trypsin Quality on MS-Based Proteomics. J. Proteomics 2012, 75 (4), 1454−1462. (6) Schilling, O.; Barré, O.; Huesgen, P. F.; Overall, C. M. Proteome-Wide Analysis of Protein Carboxy Termini: C Terminomics. Nat. Methods 2010, 7 (7), 508−511. (7) Van Damme, P.; Staes, A.; Bronsoms, S.; Helsens, K.; Colaert, N.; Timmerman, E.; Aviles, F. X.; Vandekerckhove, J.; Gevaert, K. Complementary Positional Proteomics for Screening Substrates of Endo- and Exoproteases. Nat. Methods 2010, 7 (7), 512−515. (8) Helbig, A. O.; Gauci, S.; Raijmakers, R.; van Breukelen, B.; Slijper, M.; Mohammed, S.; Heck, A. J. R. Profiling of N-Acetylated Protein Termini Provides in-Depth Insights into the N-Terminal Nature of the Proteome. Mol. Cell. Proteomics 2010, 9 (5), 928−939. (9) Xu, G.; Shin, S. B. Y.; Jaffrey, S. R. Chemoenzymatic Labeling of Protein C-Termini for Positive Selection of C-Terminal Peptides. ACS Chem. Biol. 2011, 6 (10), 1015−1020. (10) Du, X.; Fang, C.; Yang, L.; Bao, H.; Zhang, L.; Yan, G.; Lu, H. In-Depth Analysis of C Terminomes Based on LysC Digestion and Site-Selective Dimethylation. Anal. Chem. 2019, 91 (10), 6498−6506. (11) Somasundaram, P.; Koudelka, T.; Linke, D.; Tholey, A. CTerminal Charge-Reversal Derivatization and Parallel Use of Multiple Proteases Facilitates Identification of Protein C-Termini by CTerminomics. J. Proteome Res. 2016, 15 (4), 1369−1378. (12) Schnölzer, M.; Jedrzejewski, P.; Lehmann, W. D. ProteaseCatalyzed Incorporation of 18O into Peptide Fragments and Its Application for Protein Sequencing by Electrospray and MatrixAssisted Laser Desorption/Ionization Mass Spectrometry. Electrophoresis 1996, 17 (5), 945−953. (13) Kosaka, T.; Takazawa, T.; Nakamura, T. Identification and CTerminal Characterization of Proteins from Two-Dimensional Polyacrylamide Gels by a Combination of Isotopic Labeling and 2963


Article

Journal of Proteome Research Enrichment for In-Depth N-Terminome Analysis. Anal. Chem. 2016, 88 (17), 8390−8395.

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Combination of SCX Fractionation and Charge-Reversal

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