Strong Cation Exchange-based Fractionation of Lys-N-generated Peptides Facilitates the Targeted...

Strong Cation Exchange-based Fractionationof Lys-N-generated Peptides Facilitates theTargeted Analysis of Post-translationalModifications*Nadia Taouatas‡, A. F. Maarten Altelaar‡, Madalina M. Drugan, Andreas O. Helbig,Shabaz Mohammed§, and Albert J. R. Heck§

In proteomics multi-dimensional fractionation techniquesare widely used to reduce the complexity of peptide mix-tures subjected to mass spectrometric analysis. Here, wedescribe the sequential use of strong cation exchangeand reversed phase liquid chromatography in the separa-tion of peptides generated by a relatively little exploredmetallo-endopeptidase with Lys-N cleavage specificity.When such proteolytic peptides are subjected to low-pHstrong cation exchange we obtain fractionation profiles inwhich peptides from different functional categories arewell separated. The four categories we distinguish andare able to separate to near completion are (I) acetylatedN-terminal peptides; (II) singly phosphorylated peptidescontaining a single basic (Lys) residue; (III) peptides con-taining a single basic (Lys) residue; and (IV) peptides con-taining more than one basic residue. Analyzing these pep-tides by LC-MS/MS using an ion trap with both collision aswell as electron transfer-induced dissociation providesunique optimal targeted strategies for proteome analysis.The acetylated peptides in category I can be identifiedconfidently by both CID and ETcaD, whereby the ETcaDspectra are dominated by sequence informative Z-ion se-ries. For the phosphorylated peptides in category II andthe “normal” single Lys containing peptides in category IIIETcaD provides unique straightforward sequence laddersof c�-ions, from which the exact location of possible phos-phorylation sites can be easily determined. The later frac-tions, category IV, require analysis by both ETcaD andCID, where it is shown that electron transfer dissociationperforms relatively well for these multiple basic residuescontaining peptides, as is expected. We argue that thewell resolved separation of functional categories of pep-tides observed is characteristic for Lys-N-generated pep-tides. Overall, the combination of Lys-N proteolysis,low-pH strong cation exchange, and reversed phase sep-aration, with CID and ETD induced fragmentation, adds a

new very powerful method to the toolbox of proteomicanalyses. Molecular & Cellular Proteomics 8:190–200,2009.

The enormous complexity of the proteome poses a consid-erable analytical challenge for global protein identification. Anadditional order of complexity is caused by protein post-translational modifications of which many different variantsare known (1). Mass spectrometry (MS)1 is, nowadays, rou-tinely used for such complex protein identification and char-acterization studies (2, 3). Common practice in high through-put global proteome analyses is to start by proteolyticcleavage of all proteins using, most often, trypsin. The soformed peptides are then separated by reversed phase (RP)nanoflow liquid chromatography (nanoLC) and subjected totandem MS sequencing with CID as the preferred fragmenta-tion method (3). Protein identification is then accomplished bysearching the acquired peptide fragmentation data againstlarge protein sequence databases, which is greatly aided bythe availability of sequenced genomes (4). The above de-scribed general procedure for proteomics analysis provides afar from comprehensive view of the proteome and leavesroom for improvement in areas such as proteolytic cleavage(5, 6), peptide enrichment and fractionation (7–18), and pep-tide activation and fragmentation inside the mass spectro-meter (19–22). More specifically, smart combinations of pro-teases, peptide separations, and peptide fragmentationtechniques may allow the targeted analysis of specific func-tional groups of peptides, enabling the analysis of low abun-dant proteins and peptides.

Recently, electron transfer dissociation (ETD) of peptideswas introduced as an alternative peptide fragmentation

From the Biomolecular Mass Spectrometry and Proteomics Group,Bijvoet Center for Biomolecular Research and Utrecht Institute for Phar-maceutical Sciences, Utrecht University, and the Netherlands Proteom-ics Centre, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands

Received, June 23, 2008, and in revised form, September 17, 2008Published, MCP Papers in Press, September 29, 2008, DOI

10.1074/mcp.M800285-MCP200

1 The abbreviations used are: MS, mass spectrometry; SCX, strongcation exchange; RP, reversed phase; nanoLC, nanoflow liquid chro-matography; ETD, electron transfer dissociation; ESI, electrosprayionization; ETcaD, electron transfer collisional-activated dissociation;HEK, human embryonic kidney; HPLC, high pressure liquid chroma-tography; ACN, acetonitrile; LC-MS/MS, liquid chromatography tan-dem mass spectrometry; FA, formic acid; CID, collision induceddissociation.

Research

© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.190 Molecular & Cellular Proteomics 8.?This paper is available on line at http://www.mcponline.org

method. ETD cleaves peptides at the N-C� bond producingc�- and z-type ions (23–25). From the early work it is clear thatETD can be complementary to CID because it prefers largerand more basic peptides, which attain multiple charges duringelectrospray ionization (ESI) (26, 27). Reduced fragmentationefficiency of ETD for doubly charged peptides is compen-sated by the use of supplemental collisional activation(ETcaD) (28). Interestingly, ETD (and ETcaD) leaves post-translational modifications largely intact on the peptide back-bone during fragmentation thus providing, potentially, simplerspectra in which the site of modification can be easily anno-tated (29). Recently, we explored and introduced the use of arelatively little known metallo-endopeptidase for digestion ofproteins in combination with ETD (30). This metallo-endopep-tidase, termed Lys-N, has enzymatic cleavage specificity forlysine residues with cleavage occurring at the N-terminal side(31, 32). Lys-N was shown to be as sensitive and selective ascurrently used proteases and can be used for both in-gel andin-solution digestion experiments (30). Our preliminary dataindicated that the resulting proteolytic peptides are favorablefor ETD sequencing, with respect to peptide size and thenumber of charges after ESI, with the added advantage of thelysine residue being situated at the peptide N terminus. Alarge proportion of these proteolytic Lys-N peptides do notcontain any other basic residue leaving only two basic entitieseach residing at the N terminus. We argued and demonstratedthat the strong basic nature of the N-terminal side of Lys-Nproteolytic peptides attracts the proton providing the finalfragment ion charge before/after electron transfer, causingthe observed fragments to be almost exclusively c�-type ions.Utilizing this unique fragmentation behavior of Lys-N peptidesunder ETcaD conditions could markedly reduce the depend-ence on sequenced genomes and open up a complete newwindow for de novo sequencing and potentially the analysis ofpost-translational modifications in a facile manner (30). In theaccompanying paper we show that this unique feature is notsolely a characteristic of ETD fragmentation, as the samecategory of peptides provides straightforward peptide se-quence ladders in MALDI-CID-MS/MS fragmentation.2

Here, we further explore the potential of this protease inproteomics, focusing on the analysis of post-translationalmodifications. We developed a method for global proteinanalysis of whole cell lysates using a combination of Lys-Nproteolytic cleavage followed by low-pH strong cation ex-change (SCX) fractionation and RP-nanoLC-ETcaD-MS anal-ysis and RP-nanoLC-CID-MS analysis. Low-pH SCX chroma-tography has shown to be a valuable tool for phosphopeptideenrichment after tryptic digestion, although often they stillco-elute with (more abundant) acidic and N-acetylated pep-tides (8, 34, 35). We show that the combination of Lys-N and

low-pH SCX is an ideal combination for global proteome andphosphoproteome analysis, as well as for the selective en-richment and analysis of protein N-terminal peptides (36, 37).We argue that the benefits of this approach are multiplebecause we take full advantage of the proteolytic peptideproperties after Lys-N digestion in both SCX enrichment andETcaD analysis.

EXPERIMENTAL PROCEDURES

Materials—Protease inhibitor mixture was obtained from RocheDiagnostics. Metallo-endopeptidase from Grifola Frondosa (Lys-N)was obtained from Seikagaku Corporation (Tokyo, Japan). Iodoacet-amide and sodium orthovanadate were obtained from Sigma-Aldrich.DL-Dithiothreitol was obtained from Fluka Biochemical (Steinheim,Germany). Human embryonic kidney (HEK) 293 cells were providedby Dr. Pantelis Hatzis and Dr. Tokameh Mahmoudi from the Nether-lands Institute for Developmental Biology, Hubrecht Institute, TheNetherlands. HPLC-S gradient grade acetonitrile was purchased fromBiosolve (Valkenwaard, The Netherlands). Acetic acid was obtainedby MERCK KGaA (Damstadt, Germany) and high purity water ob-tained from Milli-Q system (Millipore, Bedford, MA).

HEK 293 Cells—Approximately 6 � 107 HEK 293 cells were har-vested by centrifugation for 5 min at 1200 rpm and after removal ofthe medium, cells were resuspended in 50 mM ammonium bicarbon-ate containing 25 mM sodium phosphate, 1 mM potassium fluorideand 1 mM sodium orthovanadate. After centrifugation at 1200 rpmand removal of the supernatant ice-cold lysis buffer (50 mM ammo-nium bicarbonate containing 8 M urea, protease inhibitor, 5 mM so-dium phosphate, and 1 M potassium fluoride and 1 M sodium or-thovanadate) was added to the cell pellet and the cells were lysed onice for 30 min. Subsequently, centrifugation at 20000 � g in a tabletopcentrifuge (Eppendorf, Hamburg, Germany) at 4 °C separated thesoluble and insoluble protein fractions. The soluble fraction was col-lected and the protein concentration was determined by a Bradfordassay to be 12.3 mg.

Lysate In-solution Digestion—One mg of lysate was reduced with45 mM dithiothreitol (50 °C, 15 min) followed by alkylation using 100mM Iodoacetamide (dark, RT, 15 min) and digested with Lys-N. Lys-Nwas added at a ratio of 1:85 (w/w) and the sample was incubated overnight for 37 °C. The digest was dried in a vacuum centrifuge andresuspended in 0.05% formic acid (FA).

SCX Chromatography—SCX was performed using an Agilent 1100HPLC system (Agilent Technologies) with two C18 Opti-Lynx (Opti-mized Technologies, Oregon OR) guard columns and a polysulfoethylA SCX column (PolyLC, Columbia, MD; 200 mm � 2.1 mm innerdiameter, 5 �m, 200-Å). The digested cell lysate was dissolved in0.05% formic acid, and 750 �g was loaded onto the guard column at100 �l/min and subsequently eluted onto the SCX column with 80%acetonitrile (ACN) and 0.05% formic acid. SCX buffer A was made of5 mM KH2PO4, 30% ACN and 0.05% FA, pH 2.7; SCX buffer Bconsisted of 350 mM KCL, 5 mM KH2PO4, 30% ACN and 0.05% FA,pH 2.7. The gradient was performed as follows: 0% B for 10 min,0–85% B in 35 min, 85–100% B in 6 min and 100% B for 4 min. Atotal of 49 fractions were collected and dried in a vacuum centrifuge.

ETD Experiments—The 49 dried SCX fractions were diluted in 100�l of 10% formic acid, and 1/20 (5 �l) of all the SCX fractions weresubjected to nanoscale liquid chromatography tandem mass spec-trometry (nanoLC-MS/MS) analysis, performed on an Agilent 1100HPLC system (Agilent technologies) connected to a LTQ XL Linear IonTrap Mass Spectrometer with an ETD source at the back fromThermo Fisher Scientific, Inc. (Waltham, MA).

The instrument was equipped with a 20 mm � 100 �m innerdiameter. Aqua C18 trap column (Phenomenex, Torrance, CA) and a

2 Boersema, P. J., Taouatas, N., Altelaar, A. F. M., Gouw, J. W.,Ross, L. R., Pappin, D. J., Heck, A. J. R., and Mohammed, S. (2009)Mol. Cell. Proteomics in press.

SCX-based Fractionation of Lys-N-generated Peptides

Molecular & Cellular Proteomics 8.? 191

200 mm � 50 �m inner diameter Reprosil C18 RP analytical column(Dr. Maisch, Ammerbuch-Entringen, Germany). Trapping was per-formed at a flow of 5 �l/min for 10 min, and the fractions were elutedusing a 75-min linear gradient from 0 to 40% solvent B (0.1 M aceticacid in 80% ACN (v/v), in which solvent A was 0.1 M acetic acid), 40to 100% solvent B in 2 min, and 100% B for 2.5 min. The flow ratefrom the LC was passively split from 0.360 ml/min to 100 nl/min. Thecolumn effluent was directly introduced into the ESI source of the MSusing a standard coated fused silica emitter (New Objective, Woburn,MA) (outer diameter 360 �m, tip inner diameter 10 �m) biased to 1.7kV. The mass spectrometer was operated in positive ion mode, from350 to 1500 m/z in MS mode, and with an automatic gain controlvalue of 1.00e � 05 and a max injection time of 50 ms. Parent ionswere isolated for a more accurate measurement by performing asingle ion monitoring scan and fragmented by CID and ETD in data-dependent mode with an automatic gain control value of 3.00e � 04and a max injection time of 500 ms. Ions were fragmented using CIDwith normalized collision energy of 35 and 30 ms activation time. ETDfragmentation was performed with supplemental activation; fluoran-thene was used as reagent anion, and ion/ion reaction in the ion trapwas taking place for 100 ms.

Protein Identification—Raw MS data were converted to peak listsusing Bioworks Browser software, version 3.1.1. Spectra weresearched against the International Protein Index Human databaseversion 3.36 (69012 sequences; 29002682 residues) using Mascotsoftware version 2.2.0, with Lys-N cleavage specificity. The databasesearch was made with the following parameters set to consider apeptide tolerance of �0.5 Da, a fragment tolerance of �0.6 Da,allowing two missed cleavages, Carbamidomethyl (C) as fixed mod-ification, Oxidation (M), Phosphorylation (ST), Phosphorylation (Y),and Acetylation (N-terminal) as variable modifications. Tandem massspectra assigned with a Mascot Score �30 (p value �0.05) wereaccepted providing a false discovery rate for the CID data of 4.68%and 2.53% for ETD, determined using a decoy database. Mascotinterpretation was accepted for N-terminal acetylation and phospho-rylation site assignment. All data are stored in the public depositoryPRIDE under accession numbers, 3746–3843, and the project de-scription: “A simple strategy for straightforward proteome analysis bya combination of Lys-N, strong cation exchange and electron transferdissociation”.

Lys-N Fragment Ion Statistics—For the calculation of the frequencyof occurrence of c- and z-type ions ETD MS/MS fragmentation spec-tra of unique peptides with a minimum Mascot Score of 30 and witha Lysine at the N terminus (this does not apply for N-acetylatedpeptides) were considered. The Mascot identification of the spectrawas used for automated peak fragment ion counting. For ETD spec-tra, exceptions occurred when c- or z-type ions (or related ions withammonia/water losses) were assigned to the same isotope cluster inwhich case the most appropriate assignment (e.g. based on massaccuracy trend, mono isotopic peak etc) was chosen in an automatedfashion.

RESULTS

We explored a method for global protein analysis of wholecell lysates using a combination of Lys-N proteolytic cleavagefollowed by low-pH SCX fractionation and RP-nanoLC-MS/MS analysis using both CID and ETcaD. After in-solutiondigestion of a whole HEK 293 cell lysate with Lys-N, theresulting peptides were separated into 49 fractions using alow-pH SCX separation. SCX chromatography separatespeptides primarily based on their charge state in-solution,which is governed by the protonation and deprotonation of

the basic and acidic residues, and thus the pH of the solution.We hypothesized that low-pH SCX chromatography of Lys-Nproteolytic peptides would be an ideal tool for the separationand enrichment of acetylated protein N termini, phosphoryl-ated and unphosphorylated single lysine containing peptides,as illustrated schematically in Fig. 1. Many of the Lys-N-generated peptides will contain a single basic lysine residue atthe N terminus of the peptides and therefore carry two posi-tive charges in solution (30). Similarly, a significant number ofthe Lys-N-generated peptides with acetylated N termini (i.e.mostly protein N termini) will not contain any basic group andtherefore be uncharged. Singly phosphorylated Lys-N pep-tides with no additional basic residues will carry a singlecharge in solution and should thus potentially be separatedfrom the unphosphorylated single lysine containing peptidesand, more importantly, from the acetylated protein N-terminalproteolytic peptides described above. Naturally, in the finalSCX fractions peptides will be found that contain multiplebasic residues, for instance because of the presence of argi-nine and histidine or more than one lysine residues because ofmiss-cleavages. This clear separation as represented in Fig. 1will allow prior knowledge of each fraction composition (i.e.“peptide must be N-terminally acetylated” or “peptide mustcontain a single lysine and one phosphorylated residue”) thatcan be used in the database analysis. This can lead to theremoval of certain peptides that are false positives leavingbehind a dataset with a higher overall confidence.

We hypothesized that ionization of peptides from the earlyeluting SCX fractions by ESI could be less efficient because ofthe lack of potential basic protonation sites. If these peptideswould attain just a single charge in the ESI process, analysiswith ETD would not be possible because ion-neutralizationwill occur by the invoked charge reduction. To probe thiseffect we decided to conduct alternating ETcaD and CIDfragmentation experiments on the same precursors. Each ofthe 49 SCX fractions was analyzed using RP-nanoLC-MS/MSusing in each single run alternating ETcaD and CID activationand fragmentation. The resulting spectra were searchedagainst the International Protein Index human database usingthe Mascot search engine, taking a Mascot Score �30 asthreshold. We classified, for each of the SCX fractions, allidentified peptides into 8 different categories, based on thenumber of basic residues (from 1 to 6) present in the peptide.Additionally, categories were defined for acetylated N-termi-nal peptides and phosphorylated peptides containing only asingle lysine residue.

As can be seen in Fig. 2, low-pH SCX separation of theLys-N-digested peptides results in a clear fractionation of allthese above-mentioned categories of peptides. Specifically,the enrichment of N-acetylated and singly phosphorylatedpeptides is outstanding. Nearly all of the acetylated N-termi-nal peptides are distinctly separated from the singly phospho-rylated peptides, both for the peptides identified by ETD (Fig.2A) and CID (B). Statistical analysis of the data (Fig. 2) show


192 Molecular & Cellular Proteomics 8.?

that the fractions 7 till 18 contain predominantly acetylatedprotein N-terminal peptides (92% in ETD and 80% in CID) andonly a very small number of phosphopeptides (4% in bothETD and CID). The following 8 SCX fractions (19–27) containonly �3% acetylated protein N-terminal peptides comparedwith more than 75% singly phosphorylated peptides, illustrat-ing nicely the separation power of the combination of Lys-Ndigestion and low-pH SCX separation. However, in the laterSCX fractions (24–27) also several non-phosphorylated pep-tides were identified, which predominantly contained just asingle lysine basic residue. Closer inspection of the fractionscontaining the N-acetylated protein termini resulted in theobservation that most of these peptides contained a singlebasic residue. The acetylated N-terminal peptides withoutbasic residues are present in the very early fractions (i.e. 4–6)whereas the following fractions contain almost exclusivelypeptides with one basic residue. This observation explainswhy we identify so many of the N-terminal peptides in the ETDexperiment, as without this basic residue ionization and ETDfragmentation would be hampered. It does however raise thequestion why these peptides are separated from the singlelysine-containing phosphorylated peptides, as both thesegroups of peptides should have a single charge in solution.We believe that the unique combination of N-terminal lysinepeptides and low-pH SCX conditions might play an importantrole. Although the phosphorylated single lysine peptideshave an overall charge of 1� in solution, the N-terminal endis still doubly charged and thus retention in the SCX column

might be stronger than for the single charge obtained fromthe sole basic moiety found on N-terminal acetylated pep-tides. The number of N-acetylated and phosphorylated pep-tides in the remaining later SCX fractions were also calcu-lated, but were of low frequency (see Fig. 2).

The general trends observed for ETD and CID were similarbut there were also some significant differences observed, ascan be seen in Fig. 2, A and B. CID identified significantlymore acetylated peptides in the same SCX fractions whencompared with ETD (Fig. 2B); over 2-fold more for the frac-tions 7–18. However, the vast majority of the identified N-acetylated peptides in the CID experiments had precursorpeptide ions that were doubly charged, making them, poten-tially, amenable to ETD analysis. Therefore, the relative in-creased identification efficiency observed here in the CIDexperiments, compared with ETD, likely points toward theincreased efficiency and more confident identification of thedata by Mascot for doubly charged peptides, in agreementwith published data (19, 26, 27). When inspecting the ETcaDtandem mass spectra, we observed that the majority of thedoubly charged acetylated protein N-terminal peptide precur-sor ions lead to clear MS/MS spectra mainly consisting ofz-fragment ions (as illustrated in Fig. 3, A and C). We believethat this preference for the formation of z.-ions is caused bythe acetylation of the N terminus and the presence of a basicresidue close to the C terminus, which directs the remainingproton to the C terminus. In agreement with this hypothesis,N-acetylated peptides with no basic residues lead to the

Increasing salt gradient

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FIG. 1. Expected SCX separationscheme for peptides from a Lys-N di-gest. The horizontal axis shows an in-crease in salt concentration in the elutedsample, and the vertical axis shows anincrease in intensity of the eluted peptidefractions. The uncharged Lys-N pep-tides are by theory expected to elute firstfrom an SCX column and thereafter thesingly charged Lys-N peptides and soforth. Using Lys-N, acetylated, andphosphorylated peptides should be sep-arated on the SCX column, as they pos-sess different charge states in-solution.



abundant formation of both c�- and z.-fragment ions (seesupplemental dataset present in PRIDE, accession numbers3746–3843). As demonstrated in Fig. 4, the dominant pres-ence of Z-ions is consistent over the whole range of SCXfractions from 9 to17, containing primarily the N-acetylatedprotein N termini with a single basic residue. In Fig. 3 (B andD), the associated CID spectra of the same peptides areshown. Although the CID spectra are more fragment ion-rich,they do provide a less clear picture of the peptide sequencewhen compared with the ETD spectra given in Fig. 3, A and C.

Elution of phosphorylated peptides in SCX overlaps mar-ginally with peptides containing a single lysine residue and nopost-translational modifications, as can be seen in Fig. 2. Thephosphorylated peptides were mainly observed as doublycharged ions after ESI and contained exclusively a singleN-terminal lysine as the only basic residue. ETcaD fragmen-tation of these doubly charged phosphorylated peptides re-sults in “clean” straightforward interpretable spectra consist-ing predominantly of c�-type ions, as observed and reportedpreviously for their non-phosphorylated counterparts (30). Uti-lizing Mascot annotation we calculated the frequency of thevarious fragment ions observed, and we found that over 90%of the ions were N-terminal in nature (i.e. c-type ions), as

illustrated in Fig. 4. Interestingly, these ETcaD spectra requirevery little effort to interpret because clear sequence laddersare formed with the phosphor group left intact on the peptidebackbone (Fig. 5), facilitating site-specific phosphopeptideidentification. As can be seen in Fig. 5D, tyrosine phospho-rylation could be easily determined from the sequence lad-ders. The single lysine-containing peptides are strongly en-riched in mainly three SCX fractions (28–30), which containalmost 85% of the single lysine peptide population (Fig. 2),calculated from both the ETcaD and CID analyses. In agree-ment with our earlier reported data (30) ETD analysis of thesefractions resulted in MS/MS spectra almost exclusively con-sisting of c-type ions in which c�-ions dominate, providingvery simple to interpret sequence ladders (Fig. 6A). For com-parison, in Fig. 6B is shown the complementary CID spectrumof the same precursor peptide, which reveals again a rich, butless clear fragmentation spectrum.

In the ETcaD experiments the number of generated c-typeions decreases as the charge state increases; however, stillmore than 65% of the ions observed in the fractions contain-ing peptides with three or more basic residues are c-type ions(see supplemental dataset present in PRIDE accession num-bers, 3746–3843). Here, the relative contribution of c-type

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ions is determined by the position of the additional basicresidue, i.e. an additional lysine, arginine, or histidine. If theextra basic residue is positioned close to the C terminus bothc�- and z.-ions will be formed, but when the basic residue iscloser to the N-terminal site c�-ions are dominant, as illus-trated by some examples in Fig. 6. Obviously, these highercharged peptides are still very useful in the analysis of thewhole cell lysate because ETD efficiency increases with in-creasing charge state. When comparing the global SCX pro-

files of the ETcaD and the CID experiments, CID performsrelatively well in the SCX fractions that contain peptides withup to maximally two basic residues. For peptides containingmore than two basic residues, which attain more charges inthe ESI process, a rapid decrease in the absolute and relativenumber of peptides identified by CID is observed. For thesepeptides containing multiple basic residues ETcaD performsrelatively better largely because of the increased efficiency ofthe electron transfer process. We believe that in the current

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FIG. 3. ETcaD and CID MS/MS spectra of N-terminally acetylated doubly charged ions originated from a Lys-N digest of a HEK 293cell lysate. A, ETcaD spectrum of the N-terminal acetylated peptide AcATTATMATSGSAR (m/z: 642.23, 2�) from EIF4A3 (eukaryotic initiationfactor 4A-III). A clear sequence of almost all the Z-ions are being generated because of the basic residue (R) at the C-terminal. B, CID spectrumof the N-terminal acetylated peptide AcATTATMATSGSAR (m/z: 642.16, 2�). Both b- and y-type ions are generated in the CID fragmentationprocess. C, ETcaD spectrum of the N-terminal acetylated peptide AcAAGTLYTYPENWRAF (m/z: 901.26, 2�) from EEF1G (elongation factor1-gamma). Almost a whole series of Z-ions is being generated because of the basic residue (R) close positioned to the C-terminal. TwoN-terminally c�-ions (c13 and c14) are generated from the position of the arginine residue. D, CID spectrum of the N-terminal acetylated peptideAcAAGTLYTYPENWRAF (m/z: 901.26, 2�). An almost equal amount of b- and y-ions are formed.

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FIG. 4. Occurrence (%) of c�- and z-fragment ions in the ETcaD spectra ofthe doubly charged peptides per SCXfraction. The dark gray color representsthe c�-ions and the light gray the Z-ions.Fractions 7–17 contain a lot of N-termi-nal acetylated peptides, which results inmainly the generation of Z-ions (lightgray) in ETD, whereas phosphorylatedpeptides and non-phosphorylated pep-tides with a single lysine at the N termi-nus (fraction 18–30) results in the gener-ation of almost only c�-ions (dark gray).



experimental set-up the number of peptides identified byETcaD in the lower charged fractions is relatively somewhatunderrepresented because of the suboptimal weighting in thesearch algorithm for the “unique” c�-fragment ion ladder se-quences observed for the single lysine peptides. Improvingthe ion scoring algorithms for these peptides might improvethe number of identifications.

DISCUSSION

Generally the first step in a typical global proteomics anal-ysis workflow is fractionation of a tryptic digest to reduce thecomplexity of the peptide mixtures subjected to RP-nanoLC-MS/MS. The reduction of complexity is vital to the outcome ofthe experiment because existing mass spectrometers lack thespeed to be able to sequence every peptide of a complexsample eluting from the LC column. Fractionation is oftenaccomplished by SCX chromatography of peptides formed byin-solution digestion. An additional benefit of SCX separationis the enrichment into distinct fractions of similar peptideproperties before MS analysis. This advantage can be furtherexploited by performing the SCX separation at low pH, whereafter tryptic digestion most of the phosphopeptides are foundin the early fractions of the SCX separation because of their

reduced charge states in solution because glutamates andaspartates will be neutralized and the phosphate group willpossess a negative charge (8, 34). Here, we show the addedvalue of using the metallo-endopeptidase Lys-N to digest theproteins into peptides in combination with low-pH SCX frac-tionation and ETcaD-MS and CID-MS analysis. Digestion withLys-N yields proteolytic peptides with the lysine at their Nterminus, resulting in an increased basic entity caused by thelysine and N-terminal amines. In the subsequent low-pH SCXseparation distinct fractionation profiles could be observed inwhich peptides from different functional categories were ex-tremely well separated. The four categories we were able toseparate well are (I) acetylated N-terminal peptides, (II) singlyphosphorylated peptides containing a single basic (Lys) res-idue, (III) peptides containing a single basic (Lys) residue, and(IV) peptides containing more than one basic residue. Be-cause the SCX gradient was optimized for the enrichment ofacetylated protein N-termini and phosphorylated peptides,the separation of the later eluting multiple charged peptides(i.e. containing more than one basic residue) was somewhatcompromised. Still, with the current SCX set-up the singlelysine containing peptides elute in a very clean pool spreadonly over mainly three fractions. The enrichment of post-

100 200 300 400 500 600 700 800 900 1000 1100m/z

200 300 400 500 600 700 800 900 1000 1100m/z

c2

(M+2H) ++

c6

c5 c4

c7

c8

c3

c9

b2 y3-98

b3 b8-98

b9-98

b6

b7

b5

y5-98

y04-98

b4 y4-98

200 400 600 800 1000 1200 1400 1600 1800m/z

c4c3

c5c6

(M+2H) ++

c7

c8c9

c10 c11

c12

c13

c14K N S S L L pSpS F D N E D E N E

Score: 75

200 400 600 800 1000 1200m/z

(M+2H) ++

c3c4

c5

c6 c7c8

c9

c10

K I G E G T pYpY G V V Y

Score: 68

pY

pS

pS

K L T G pS pS T S S L N

Score: 66

K L T G pSpS T S S L N

Score: 84

A ETcaD B

C DETcaD ETcaD

CID

FIG. 5. ETcaD and CID MS/MS spectra of phosphorylated doubly charged ions originating from Lys-N generated peptides from HEK293 cells. A, ETcaD MS/MS spectrum of serine phosphorylated KLTGpSTSSLN (m/z: 544.11, 2�) from EXOC1 Isoform 1 of Exocyst complexcomponent 1. From the sequence of c�-ions it is easy to determine the phosphorylation site. B, CID MS/MS spectrum of serine phosphorylatedKLTGpSTSSLN (m/z: 544.08, 2�). Compared with the ETcaD spectrum a clear sequence for interpretation of the phosphorylation site is notobserved. C, ETcaD MS/MS spectrum of serine phosphorylated KNSSLLpSFDNEDENE (m/z: 910.68, 2�) from the uncharacterized proteinENSP00000307425. Nearly the entire sequence is fragmented into c�-ions by the ETcaD process. The phosphate group is not lost in thefragmentation process. D, ETcaD MS/MS spectrum of tyrosine phosphorylated KIGEGTpYGVVY (m/z: 633.13, 2�) CDK3 Cell division proteinkinase 3. By interpretation of the peptide fragment ions in the spectrum the sequence and site of phosphorylation can be determined, as onlyc�-ions are generated, and the phosphate group is not lost in the fragmentation process.



translational modified peptides is relatively high in our currentexperiments when taking into account that no additional en-richment method is applied and that solely in-solution chargestate separation has been used.

Most interestingly the proposed set-up results in a fraction-ation of peptides whereby there is almost no overlap betweenthe N-acetylated and phosphorylated peptides. N-terminalacetylation, and less so propionylation, of proteins is a com-mon and important process in cellular biology, which is forinstance linked to protein stability, protecting the proteins

from attack by aminopeptidases (35, 38–40). Because �90%of cellular proteins in eukaryotic cells contain blocked N ter-mini, they make up a significant portion of the peptide pooland are of great interest with respect to the examination ofN-terminal protein processing or alternatively terminated pro-tein isoforms (33, 35). Our data show that the Lys-N proteo-lyzed acetylated protein N termini in category I form primarilyz�-ions in ETcaD fragmentation (�70%; Figs. 3 and 4) causedby the blockage of the N terminus and the presence of a basicresidue.

400 600 800 1000 1200 1400 1600 1800 2000m/z

b14

b15

b13

b16y17y16

b12

y11

b11

b8

b9 y11

y7y5

b10y6

y8

y4b7

b6b5y13

y12

200 400 600 800 1000 1200 1400 1600 1800 2000

(M+2H) ++

c4 c5c7

c8c11 c12

c13c18

c16

c15c14

c17

c9c6

c10

m/z

B

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

(M+2H) ++

c4 c6 c7 c8c9

c10

c11c5c13

c15

c16

c14

c12

200 400 600 800 1000 1200 1400 1600 1800m/z

c6c4

c3

c2

(M+2H) ++

(M+3H) +++

c5

z11 z12

z15z16z14z13

c10c9c7z8

z10

c14c17

c16c15c12 c13c8

z9

(M+3H) +++

200 400 600 800 1000 1200 1400 1600m/z

z2c2 c3c4

z3 z4 z5 z6

c5c6

z7

c7

c9

c10

z10 c12

z9 z11c11 c14c13

z14z12 z15z13c15z8

c8

y6

y9

y10

y11

y8

400 600 800 1000 1200 1400 1600m/z

y12y13

y14y15

y2

b8

b6

b5b7y4 y5

y3 b9

b10

b11 b12 b14b13

y7

Score: 120

D

Score: 105

K N T G V I L A N D A N A E R L

C ETcaD

Score: 49

K R I Q E I I E Q L D V T T S E Y E

E ETcaD F

Score: 87

K P W A L T F S Y G R A L Q A S A L

ETcaD

CID

CID

A ETcaD

Score: 103

K Q A F D D A I A E L D T L N E D S Y

Score: 79

K Q A F D D A I A E L D T L N E D S Y

K N T G V I L A N D A N A E R L

FIG. 6. ETcaD and CID MS/MS spectra of double- and triple-charged ions originating from Lys-N-generated peptides from HEK 293 cells.A, ETcaD spectrum of doubly charged peptide KQAFDDAIAELDTLNEDSY (m/z: 1079.34, 2�) from YWHAH 14–3-3 protein eta. Protons/charges in Lys-N-generated peptides, with a single basic residue, will be preferentially located at the N terminus because of the presence oftwo free amine groups, which results in the exclusive generation of c�-type fragment ions. B, CID spectrum of doubly charged peptideKQAFDDAIAELDTLNEDSY (m/z: 1079.36, 2�) from YWHAH 14–3-3 protein eta. CID fragmentation does not result in the exclusive generationof N-terminal fragment ions. C, ETcaD spectrum of triple-charged peptide KNTGVILANDANAERL (m/z: 566.82, 3�) from NOL1 94 kDa protein.Lys-N-generated peptides, with a basic residue contiguous to the c-terminal, result in the formation of almost equally numbers of c�- andZ-ions. D, CID spectrum of doubly charged KNTGVILANDANAERL (m/z: 849.86, 2�) from NOL1 94 kDa protein. In CID the same trend is notobserved as mainly y-ions are generated. E, ETcaD spectrum of doubly charged peptide KRIQEIIEQLDVTTSEYE (m/z: 1097.45, 2�) fromHSPD1 60 kDa heat shock protein, mitochondrial precursor. Lys-N-generated peptides, with a second basic residue adjacent to the N-teminallysine, will result in the exclusive generation of c�-type fragment ions. F, ETcaD spectrum of triple-charged peptide KPWALTFSYGRALQASAL(m/z: 660.53, 3�) from ALDOA fructose-bisphosphate aldolase A. Lys-N-generated peptides, with a second basic residue in the center of thepeptide sequence, will result in the generation of c�- and z-type fragment ions.



An additional advantage of the almost complete separationis that the fractions containing acetylated protein N termini areremoved as “contamination” from the phosphorylated frac-

tions, allowing enrichment and targeted analysis of the phos-phoproteome. ETcaD analysis of the purified phosphopep-tides (category II) results in clear c�-ion ladder sequences of

FIG. 7. General overview describing the dominance of particular fragmentation patterns observed in ETcaD analysis of (A) peptides with anacetylated N terminus containing a single basic residue (Group I). B, phosphorylated peptides containing a single N-terminal lysine (Group II).C, peptides containing a single N-terminal lysine (Group III). D, peptides with at least two basic residues in their sequence (Group IV).



the peptides and allows a simple read out of the location ofthe phosphorylation. Peptides containing a single lysine andno other basic residues (category III) can be also be purifiedvia low pH SCX. These peptide ions can be preferentiallyanalyzed by ETcaD as with this activation method simplec�-ion sequence ladders are generated as illustrated in Fig.6A. These sequence ladders can potentially facilitate de novosequencing of peptides from species with unsequenced ge-nomes or unknown isoforms, circumventing the need for theavailability of databases containing known protein molecularsequences.

A general overview of the fragmentation patterns observedin ETcaD analysis of the peptides seen in the four differentcategories is shown in Fig. 7. Fig. 7A shows the preferredETcaD fragmentation of the acetylated protein N terminuspeptides (category I). Because the N terminus is blocked, theremaining proton after the ETcaD analysis will preferentiallygo to the C terminus and in the resulting spectra z�-ions willdominate. Both the phosphorylated and unphosphorylatedsingle lysine peptides (Fig. 7, B and C) (category II and III,respectively) will produce spectra dominated by c�-ions afterETcaD analysis because of the favored protonation of the Nterminus. Finally, the peptides containing multiple basic resi-dues, category IV, form both c�- and z�-ions. Here a prefer-ence for c-type ions is only observed when the basic residuesare close to the N terminus (also see Fig. 6E), and equaloccurrence of c- and z-type ions, when the basic residue isclose to the C terminus (also see Fig. 6C), as also shown fortryptic peptides with electron capture dissociation.

In summary, the use of the metallo-endopeptidase Lys-N incombination with SCX described here in detail is a powerfulproteomics method. It allows the separation to near comple-tion of (I) acetylated N-terminal peptides (with and without asingle basic residue), (II) singly phosphorylated peptides con-taining a single basic (Lys) residue, (III) peptides containing asingle basic (Lys) residue, and (IV) peptides containing morethan one basic residue. Analyzing these peptides by LC-MS/MS with both CID as well as ETcaD provides uniqueoptimal targeted strategies for proteome analysis of theseclasses of peptides. Strikingly, ETcaD provides a facilemethod for site localization of phosphorylated peptides incategory II and facilitates a database independent method forsequencing of “normal” single lysine containing peptides.Overall, the combination of the use of the Lys-N protease,with SCX and RP separation, and CID- and ETD-inducedfragmentation, adds a new very powerful method to the tool-box of proteomic analyses enabling also facile analysis ofpeptides and their post-translational modifications.

* This work was supported by the Netherlands Proteomics Centre,a program embedded in the Netherlands Genomics Initiative. Thecosts of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

‡ Both authors contributed equally to this work.§ To whom correspondence may be addressed. E-mail:

[email protected] or [email protected].

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Strong Cation Exchange-based Fractionation of Lys-N-generated Peptides Facilitates the Targeted...

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