MS-Based Quantitative

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Isobaric Peptide Termini Labeling for MS/MS-Based Quantitative Proteomics Christian J. Koehler,† Margarita Strozynski,† Frank Kozielski,‡ Achim Treumann,§ and Bernd Thiede*,† The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, 0349 Oslo, Norway, The Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow G61 1BD, Scotland, United Kingdom, and NEPAF, Devonshire Building, Newcastle upon Tyne, NE1 7RU, United Kngdom Received May 13, 2009

Since its introduction, isobaric peptide labeling has played an important role in relative quantitative comparisons of proteomes. This paper describes isobaric peptide termini labeling (IPTL), a novel approach for the identification and quantification of two differentially labeled states using MS/MS spectra. After endoproteinase Lys-C digestion, peptides were labeled at C-terminal lysine residues with either 2-methoxy-4,5-dihydro-1H-imidazole (MDHI) or with tetradeuterated MDHI-d4. Subsequently, their N-termini were derivatized either with tetradeuterated succinic anhydride (SA-d4) or with SA. The mixed isotopic labeling results in isobaric masses and provided several quantification data points per peptide. The suitability of this approach is demonstrated with MS and MS/MS analyses of Lys-C digests of standard proteins. A conceptually simple quantification strategy with a dynamic range of 25 is achieved through the use of Mascot score ratios. The utility of IPTL for the analysis of proteomes was verified by comparing the well-characterized effect of the antimitotic inhibitor S-Trityl-L-Cysteine (STLC) on HeLa cells that were treated for either 24 or 48 h with the inhibitor. Many apoptosis-linked proteins were identified as being differentially regulated, confirming the suitability of IPTL for the analysis of complex proteomes. Keywords: Apoptosis • isobaric labeling • IPTL • quantitative proteomics • S-Trityl-L-cysteine

Introduction Proteomics has undoubtedly contributed substantially to the growth of scientific knowledge over the past decade.1,2 There is now an emerging consensus that further progress in proteomics-based research must provide quantitative data. Quantitative mass spectrometry has been successfully applied to proteomics since the introduction of isotope-coded affinity tagging (ICAT).3 Principally, there are three different ways of introducing an isotopic label into a biological specimen: metabolic labeling, proteolytic labeling, or chemical derivatization. Metabolic labeling with stable isotope labeling by amino acids in cell culture (SILAC)4 requires living cells, whereas proteolytic and chemical labeling can be performed with any proteome. Proteolytic oxygen-18 labeling utilizes a proteinase and H2O18 to produce labeled peptides.5 A variety of different approaches have been established for quantitative proteomics using chemical labeling that exploit generally either the reactivity of thiol groups of cysteines or of primary amine groups on lysine residues or the N-termini of peptides.6 ICAT consists of a cysteine-directed reactive group, a linker region including * To whom correspondence should be addressed. The Biotechnology Centre of Oslo, University of Oslo, P.O. Box 1125 Blindern, 0317 Oslo, Norway. Tel.: +47-22840533. Fax: +47-22840501. E-mail: [email protected]. † University of Oslo. ‡ The Beatson Institute for Cancer Research. § NEPAF. 10.1021/pr900425n CCC: $40.75

 2009 American Chemical Society

different stable isotopes, and a biotin-affinity tag for purification.3 The ICAT approach reduces the complexity of the sample, but is limited to proteins containing cysteines. By contrast, quantitative labeling of amine groups allowed the quantification of all peptides after proteolytic digestion and was named global internal standard technology (GIST).7 Furthermore, isotope-coded protein labeling (ICPL) was established for chemical isotopic labeling of proteins on lysines.8 An important drawback of isotopic labeling is an increase in sample complexity. Generally, the number of peaks in an LC/MS chromatogram is at least doubled, aggravating already difficult issues related to sampling of peptides for identification purposes. This problem was successfully addressed with the isobaric labeling strategies such as isobaric tagging for relative and absolute quantification (iTRAQ)9 and tandem mass tagging (TMT).10 These labeling reagents are composed of three different segments. The first of these segments can be of different molecular weights and generates a fragment of which the intensity can be used for quantification. The second fragment provides the balance to ensure that the reagent has the same molecular weight for a variety of different first segments. The third segment supplies a reactive group that is used to quantitatively attach the reagent to peptides. Isobaric labeling has been very successful since its first introduction. Importantly, relative quantification of up to eight different samples can be achieved simultaneously.11 However, Journal of Proteome Research 2009, 8, 4333–4341 4333 Published on Web 08/05/2009

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some drawbacks of isobaric labels have been noted. The low molecular weight of the signature ions makes it difficult to use isobaric labeling reagents with ion trap mass spectrometers due to the low molecular weight exclusion limit. Although the structure of the currently available isobaric labeling reagents has been designed to avoid the overlap of signature ions with naturally occurring peptide fragments, this is not always the case and the resulting interference is causing quantification errors. To overcome these drawbacks while retaining the advantages of isobaric labeling, we have developed a novel approach using crosswise isobaric peptide termini labeling (IPTL). The approach was established with proteins digested with endoproteinase Lys-C, producing peptides that have lysines at the C-termini. These lysine residues are then selectively modified using 2-methoxy-4,5-dihydro-1H-imidazole (MDHI), which is commercially available in two different forms, nonlabeled and as a tetradeuterated reagent (MDHI-d4).12 Subsequently, the N-termini of MDHI-derivatized peptides are reacted with tetradeuterated succinic anhydride (SA-d4) and the N-termini of MDHI-d4-derivatized peptides are reacted with succinic anhydride (SA).13 The resulting mixtures of isobaric peptides can be pooled and analyzed together. We demonstrate the effectiveness of IPTL labeling using purified proteins and validate the suitability of this strategy for the analysis of complex proteomes by monitoring changes in the proteome of HeLa cells in response to the antimitotic inhibitor S-TritylL-cysteine (STLC).

Experimental Section Cell Culture, Induction of Apoptosis, and SDS-PAGE. HeLa cells were grown as a monolayer in RPMI supplemented with 10% fetal bovine serum and maintained in a humid incubator at 37 °C in a 5% CO2 environment. Cells were treated with 5 µM S-Trityl-L-cysteine (STLC) from a 5 mg/mL stock in DMSO.14 Cells were trypsinized after 24 and 48 h, harvested, resuspended in 1 mL of PBS, and centrifuged again at 10 000 rpm. Cell pellets were frozen in liquid nitrogen and stored at -20 °C. SDS-PAGE was performed with 4% stacking gel and 10% separation gel using a Mini-Protean 3 cell (Bio-Rad, Oslo, Norway).15 Gels were stained with Coomassie Brilliant Blue G-250 (Serva, Heidelberg, Germany) employing the blue silver staining technique with slight modifications.16 Fixation was performed with 50% ethanol/2% phosphoric acid for 1 h, incubation with 34% ethanol/2% phosphoric acid/17% ammonium sulfate for 1 h, and staining with 20% methanol/10% phosphoric acid/10% ammonium sulfate for 1 h. Finally, the gels were washed once for 30 min with 25% ethanol and three times with water. In-Gel Lys-C Digestion. Coomassie G-250 stained gel lanes were cut into 12 bands with a scalpel for in-gel digestion with 0.06 µg of Lys-C (Sigma-Aldrich, Oslo, Norway) in 60 µL of 25 mM Tris, pH 8, 1 mM EDTA for 16 h at 37 °C. For each band, the Lys-C produced peptides were purified with µ-C18 ZipTips (Millipore, Billerica, MA), and dried using a Speed Vac concentrator (Savant, Holbrook, NY). In-Solution Lys-C Digestion. Fetuin (bovine), lactoglobulin (bovine), transferrin (human), myoglobin (sperm whale), and serum albumin (bovine) were purchased from Sigma-Aldrich (Oslo, Norway). The proteins were dissolved in Lys-C buffer (25 mM Tris, pH 8.5, and 1 mM EDTA) and digested with Lys-C (enzyme to protein ratio 1:50) for 16 h at 37 °C. The digestion 4334

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Figure 1. Flowchart of the IPTL approach. Proteins of two different states were digested with Lys-C to produce peptides with lysine residues (K) at the C-terminus. Next, the peptides were modified at lysines with MDHI and MDHI-d4, respectively. Subsequently, isobaric peptides were generated by crosswise derivatization of the N-termini with SA-d4 and SA, respectively. After mixing of the two samples, isobaric peptide masses were detected for peptides in MS mode. Relative quantification of the two states was achieved at the MS/MS level using the fragment pairs with 4 Da differences. b-Ions and y-ions resulted in reversed quantification values. The structures of MDHI and SA are shown in the box at the bottom.

was stopped by adding formic acid to a final concentration of 0.8%. The Lys-C produced peptides were purified with µ-C18 ZipTips (Millipore), and dried using a Speed Vac concentrator (Savant). Derivatization of Lysine Residues. 2-Methoxy-4,5-dihydro1H-imidazole (MDHI) and the tetradeuterated form 2-methoxy4,5-dihydro-1H-imidazol-4,4,5,5-d4 (MDHI-d4) of the reagent were purchased from C/D/N Isotopes (Point Claire, Quebec, Canada). An aqueous solution of 800 mM 2-methoxy-4,5dihydro-1H-imidazole (d0 and d4) was prepared and 20 µL of this solution was added to the purified and dried Lys-C digests, thoroughly mixed, and incubated for 3 h at 55 °C. The Lys-C peptides were purified with µ-C18 ZipTips (Millipore), and dried using a Speed Vac concentrator (Savant). N-Terminal Peptide Succinylation. A solution of 100 mM succinic anhydride (SA) (Sigma-Aldrich, Oslo, Norway) or tetradeuterated succinic anhydride-d4 (SA-d4) (Larodan Fine Chemicals AB, Malmo¨, Sweden) was freshly prepared in 500 µL of 200 mM sodium dihydrogenphosphate buffer and the pH was adjusted to 6.5 with 3 µL of ammonium hydroxide (30%). Twenty microliters of SA-solution was added to the purified and dried Lys-C peptide digests derivatized with MDHI-d4 or 20 µL of SA-d4-solution was added to the dried Lys-C peptide

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Figure 2. MALDI-MS and MS/MS spectra of a Lys-C peptide of BSA after IPTL. The peptide mass fingerprint confirms that IPTL derivatization ran to completion (A). Panels B-D show MS/MS spectra of the Lys-C produced BSA peptide DLGEEHFK after IPTL labeling with MDHI-d4/SA and with MDHI/SA-d4. The crosswise labeled peptides were mixed together in the ratios 1:1 (B), 2:1 (C), and 5:1 (D). The detected y-ion series occurred as doublets with 4 Da mass differences. The lower masses of these pairs corresponded to labeling with MDHI/SA-d4, and the higher masses to MDHI-d4/SA labeling. The mass range around the y2-ion at 362/366 Da was magnified to exemplify a peak pair. Neutral loss of succinic acid (NL) was observed at 1042 Da (SA-d4) and 1046 Da (SA).

digests derivatized with MDHI. After thorough mixing and incubation for 1 h at 37 °C, the peptides were purified with µ-C18 ZipTips (Millipore), and dried using a Speed Vac concentrator (Savant). For analysis by mass spectrometry, the modified Lys-C peptides were reconstituted in 5 µL of 1% formic acid and combined. MALDI-TOF/TOF-MS. An Ultraflex II (Bruker Daltonics, Bremen, Germany) MALDI-TOF/TOF mass spectrometer was used after external calibration with kemptide, bradykinin, substance P, glu-fibrinopeptide B, and dynorphin A 2-17 (Sigma-Aldrich, Oslo, Norway or Bachem, Basel, Switzerland). The samples were analyzed in the TOF mode for the generation of peptide mass fingerprints as well as in the TOF/TOF mode for fragmentation analysis of chosen peaks. R-Cyano-4-hydroxycinnamic acid (20 mg/mL) in 0.3% aqueous trifluoroacetic acid/acetonitrile (2:1) was used as matrix. The samples were applied to a ground steel sample holder and introduced into the mass spectrometer after drying. Basic settings of the MALDI-TOF/TOF instrument (Ultraflex II, Bruker Daltonics) were as follows: Ion source 1, 25 kV; ion source 2, 21.85 kV; lens, 9.60 kV; reflector, 26.3 kV; reflector 2, 13.85 kV; deflector mode, polarity positive. Mass spectra were transformed into peak lists using the SNAP algorithm of the software FlexAnalysis version 2.4 (Bruker Daltonics). NanoLC-LTQ Orbitrap Mass Spectrometry. The dried peptides were dissolved in 10 µL of 1% formic acid in water and 3 µL was injected into an Ultimate 3000 nanoLC system (Dionex, Sunnyvale CA) connected to a linear quadrupole ion trap-

orbitrap (LTQ-Orbitrap XL) mass spectrometer (ThermoScientific, Bremen, Germany) equipped with a nanoelectrospray ion source. An Acclaim PepMap 100 column (C18, 3 µm, 100 Å) (Dionex) with a capillary of 12 cm bed length was used for separation by liquid chromatography. A flow rate of 300 nL/ min was employed with a solvent gradient of 7-40% B in 45 min for the standard protein mixture and in 90 min for proteins of the STLC-treated HeLa cells. Solvent A was 0.1% formic acid, whereas aqueous 90% acetonitrile in 0.1% formic acid was used as solvent B. The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap-MS and LTQMS/MS acquisition. Survey full scan MS spectra (from m/z 300 to 2000) were acquired in the Orbitrap with resolution R ) 60 000 at m/z 400 (after accumulation to a target of 1 000 000 charges in the LTQ). The method used allowed sequential isolation of the most intense ions, up to six, depending on signal intensity, for fragmentation on the linear ion trap using collisional induced dissociation (CID) at a target value of 100 000 charges. For accurate mass measurements, the lock mass option was enabled in MS mode and the polydimethylcyclosiloxane (PCM) ions generated in the electrospray process from ambient air were used for internal recalibration during the analysis.17 Target ions already selected for MS/MS were dynamically excluded for 60 s. General mass spectrometry conditions were electrospray voltage, 1.5 kV; no sheath and auxiliary gas flow. Ion selection threshold was 500 counts for MS/MS, and an activaJournal of Proteome Research • Vol. 8, No. 9, 2009 4335

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Table 1. STLC-Modified Proteins Identified Using IPTL protein name

acc. no

score/QM (MDHI-d4/SA)

score/QM (MDHI/SA-d4)

rel. score

Polyadenylate-binding protein 4 Nucleolin T-complex protein 1 subunit eta Heat shock protein 105 kDa Stress-70 protein, mitochondrial Vimentin Pyruvate kinase isozymes M1/M2 Histone H1.2 Lamin-A/C T-complex protein 1 subunit beta Purine nucleoside phosphorylase Histone-binding protein RBBP4 Heat shock cognate 71 kDa protein T-complex protein 1 subunit delta U2 small nuclear ribonucleoprotein A′ Nuclear autoantigenic sperm protein Far upstream element-binding protein 2 Protein disulfide-isomerase Catalase Protein RCC2 Thioredoxin Malate dehydrogenase, mitochondrial 3-hydroxyisobutyryl-CoA hydrolase, mitochondrial Inosine-5′-monophosphate dehydrogenase 2 Fructose-bisphosphate aldolase A L-lactate dehydrogenase B chain Kinesin-1 heavy chain Poly (ADP-ribose) polymerase 1 3-ketoacyl-CoA thiolase, mitochondrial Stathmin Annexin A1 60S ribosomal protein L4 Nucleophosmin Medium-chain specific acyl-CoA dehydrogenase 60 kDa heat shock protein, mitochondrial Talin-1 Alpha-actinin-4 RuvB-like 1 Phosphoglycerate kinase 1 Annexin A2 Serpin H1 Interleukin enhancer-binding factor 3 Actin, cytoplasmic 1 Filamin-B Histone H4 Alpha-enolase Myosin-9 Serine/threonine-protein kinase PAK 2 S-methyl-5′-thioadenosine phosphorylase Caldesmon Src substrate cortactin ATP-dependent DNA helicase 2 subunit 2 26S proteasome non-ATPase regulatory subunit 3 Heterogeneous nuclear ribonucleoprotein M Ubiquitin-like modifier-activating enzyme 1 Targeting protein for Xklp2 Heterogeneous nuclear ribonucleoprotein Q Putative adenosylhomocysteinase 2 Proteasome subunit alpha type-1 Lysyl-tRNA synthetase ATP-binding cassette subfamily E member 1 GTP-binding nuclear protein Ran Elongation factor 1-alpha 1 Peroxiredoxin-1 Peroxiredoxin-2 ATP synthase subunit beta, mitochondrial

Q13310 P19338 Q99832 Q92598 P38646 P08670 P14618 P16403 P02545 P78371 P00491 Q09028 P11142 P50991 P09661 P49321 Q92945 P07237 P04040 Q9P258 P10599 P40926 Q6NVY1 P12268 P04075 P07195 P33176 P09874 P42765 P16949 P04083 P36578 P06748 P11310 P10809 Q9Y490 O43707 Q9Y265 P00558 P07355 P50454 Q12906 P60709 O75369 P62805 P06733 P35579 Q13177 Q13126 Q05682 Q14247 P13010 O43242 P52272 P22314 Q9ULW0 O60506 O43865 P25786 Q15046 P61221 P62826 P68104 Q06830 P32119 P06576

110/9 415/32 117/7 123/12 114/7 97/3 132/14 169/11 170/9 140/7 85/6 74/6 652/50 63/6 62/4 80/4 54/5 135/8 60/3 41/1 50/2 35/3 34/1 51/6 213/11 46/4 36/3 41/2 36/3 25/1 38/5 51/2 58/5 33/1 388/34 29/1 34/3 28/1 38/2 39/5 39/3 36/6 55/6 26/2 25/1 34/6 46/4 169/10 111/4 87/3 81/4 77/7 77/3 72/4 69/4 68/3 62/5 62/3 61/3 58/3 57/4

29/3 112/14 33/4 35/5 38/3 33/3 46/8 59/4 61/6 52/7 33/1 29/2 278/28 27/2 27/1 35/1 25/2 63/5 28/1 82/4 101/4 73/4 71/3 107/7 462/23 103/6 81/8 93/4 84/6 59/5 94/7 133/6 154/19 90/3 1064/52 101/5 131/12 117/5 165/19 173/14 176/9 163/9 316/35 159/10 203/4 311/17 734/31

3.79 3.71 3.55 3.51 3.00 2.94 2.87 2.86 2.79 2.69 2.58 2.55 2.35 2.33 2.30 2.29 2.16 2.14 2.14 0.50 0.50 0.48 0.48 0.48 0.46 0.45 0.44 0.44 0.43 0.42 0.40 0.38 0.38 0.37 0.36 0.29 0.26 0.24 0.23 0.23 0.22 0.22 0.17 0.16 0.12 0.11 0.06

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24, 25 24-27 24, 28 24 12, 24, 28, 29 24 30 14 28 31 28 24 24 29 14, 27 24 32 33

34

14, 24, 26, 27 14 35 24, 26-28

24, 26

36

24, 29, 37 30 29, 30 14, 24, 27 38 39 40

27, 30, 41

27 42 29 417/13 210/10 121/16 115/4 113/3

43 37 44 28

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Isobaric Peptide Termini Labeling Table 1. Continued protein name

acc. no

Protein DJ-1 Transgelin-2 Ras-related protein Rab-7a Heterogeneous nuclear ribonucleoprotein U Histone H2B type 1-C/E/F/G/I Nucleoprotein TPR Elongation factor 1-gamma Alpha-actinin-1 6-phosphogluconate dehydrogenase, decarboxyl. Puromycin-sensitive aminopeptidase Catenin alpha-1 Eukaryotic translation initiation factor 4 gamma 1 Fumarate hydratase, mitochondrial RuvB-like 2 Spectrin alpha chain Glyoxalase domain-containing protein 4 Plectin-1 Adenylyl cyclase-associated protein 1 High mobility group protein B2 40S ribosomal protein S7 Hepatoma-derived growth factor Histone H2B type 1-B PEST proteolytic signal-containing nuclear protein Leukotriene A-4 hydrolase Acyl-protein thioesterase 1

Q99497 P37802 P51149 Q00839 P62807 P12270 P26641 P12814 P52209 P55786 P35221 Q04637 P07954 Q9Y230 Q13813 Q9HC38 Q15149 Q01518 P26583 P62081 P51858 P33778 Q8WW12 P09960 O75608

score/QM (MDHI-d4/SA)

score/QM (MDHI/SA-d4)

111/4 111/6 108/3 101/8 98/4 86/6 84/5 82/6 79/5 78/3 77/6 73/3 68/5 63/3 62/3 61/3 58/5 55/3 55/5 55/3 53/6 51/3 51/3 50/3 50/4

rel. score

ref.

45

24 29 29 26

46 24

37 47 14 24 28 30

a The protein names, Swiss-Prot accession numbers, the Mascot protein score (score) and the number of matched queries (QM) searching the respective set of fixed modifications are displayed. Incubation of HeLa cells with STLC for 24 h to induce mitotic arrest corresponded to MDHI-d4/SA and for 48 h to trigger apoptosis to MDHI/SA-d4. The relative protein score (rel. score) was calculated if the protein was identified with both sets of fixed modifications (MDHI-d4/SA)/(MDHI/SA-d4). References (ref.) were cited showing that most of the proteins have previously been identified to be linked to apoptosis.

tion Q-value of 0.25 and activation time of 30 ms were also applied for MS/MS. Data Analysis. Raw LTQ Orbitrap XL data were processed using DTA supercharge software to generate mgf files. Then, a database search was performed by tandem mass spectrometry ion search algorithms from the Mascot in-house version 2.2.1 by database comparisons18 with mammalian (63 892 sequences) or human entries (20 411 sequences) from Swiss-Prot (20081212). Lys-C was selected as enzyme without any missed cleavage sites and tolerance of 10 ppm for the precursor ion and 0.6 Da for the MS/MS fragments was applied. Moreover, methionine oxidation was allowed as variable modification. Fixed modifications were set to the two corresponding modifications SA/MDHI-d4 or SA-d4/MDHI, respectively. Automatic decoy database searches were performed in Mascot and revealed a false discovery rate for peptide matches above an identity threshold of less than 2% for the Lys-C digested proteins of HeLa cells. Proteins were considered to be identified by Mascot if a probability 2 and 28 proteins returned a Mascot score ratio of 50 when MDHI/SA-d4 was set as fixed modifications. Conversely, 14 proteins were only identified when MDHI-d4/SA was set as the fixed modifications (with at least three peptides and a Mascot score >50), but not when MDHI/SA-d4 was set as the fixed modifications. Many of the proteins that were highlighted by this analysis were detected in a previous, two-dimensional electrophoresis-based study of STLC-induced apoptosis or in analyses of apoptotic events reported by other laboratories (Table 1). To give an example, lamin-A/C was identified with

We present a novel strategy for isobaric protein quantification based on the derivatization of peptide termini with complementary isotopically labeled reagents. We have shown that isobaric peptide termini labeling is quantitative, based on the absence of nonlabeled peptides or of peptides that are only labeled on one terminus in the peptide mass fingerprint. We have also shown that pairs of deuterium labeled IPTL-derivatized peptides do not differ in retention time on reversed phase chromatography, ensuring accurate and easy quantification from MS/MS spectra. On the other hand, dimethylation of the N-termini using formaldehyde (d0, and d2) can be used instead of succinic anhydride (d0, and d4). A straightforward, conceptually simple semiquantitative way of evaluating IPTL data is provided using Mascot score ratio values. Finally, we show that IPTL can be used for the quantification of complex proteomic changes by providing data replicating the well-documented changes of apoptotic cells upon incubation with STLC14 or other inducers of apoptosis.24-47 IPTL shares several critical advantages with other isobaric peptide quantification methods such as iTRAQ and TMT. Unlike nonisobaric quantification methods, sample complexity at the MS level is not increased, providing improved sensitivity, reproducibility and protein coverage. The important difference between IPTL and established isobaric labeling methods is the doubling of all sequence determining ions in the MS/MS spectra rather than the presence of a low molecular weight region containing quantification fragments. This has several consequences: First, IPTL spectra are better suitable for relative quantification using ion trap mass spectrometers, which are less sensitive in the low molecular weight region. Second, several quantifiable ion pairs are recorded per MS/MS spectrum, providing the option of statistical treatment of the result with increased confidence in quantification accuracy. Third, the presence of pairs of b-ions and y-ions with reverse quantification ratios increases the confidence of database hits and/or aids in the assignment of ions when de novo sequencing has been carried out. In the present study, we have used a straightforward approach for the semiquantitative evaluation of IPTL data, exploiting the effect of nonassigned fragment ions on the MOWSE score. The Mascot score ratio values (see Experimental Section) turn out to be surprisingly accurate in highlighting proteins and peptides that are relatively enriched or depleted in the data sets that we have compared. While this approach does not exploit all of the IPTL advantages that we have outlined, it is very straightforward and accessible to laboratories that do not have access to tailored software. We are, at the moment, developing IPTL-based protein quantification and identification software. We have used endoproteinase Lys-C for the proteolytic digest prior to IPTL analysis of protein mixtures. This has the advantage of decreasing the complexity of resulting peptide mixtures and of making sure that every proteolysis product bar the protein C-termini will produce a quantification signal. This Journal of Proteome Research • Vol. 8, No. 9, 2009 4339

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Figure 5. Selected MS/MS spectrum for the relative quantification of lamin-A/C during STLC-induced apoptosis. The MS/MS spectrum of the isobaric peptide EAALSTALSEK with m/z 646.34 digested with Lys-C and subsequently crosswise labeled with MDHI-d4/SA (STLC, 24 h) and MDHI/SA-d4 (STLC, 48 h) is presented. The ratios of the intensities of the detected b- and y-ions are shown in parentheses (MDHI/SA-d4 divided by MDHI-d4/SA). The ratios of the peak intensities revealed an average of 0.46 ( 0.11 and a median of 0.49 for the b-ions, and an average of 2.29 ( 0.36 and a median of 2.19 was obtained for the y-ions.

strategy could be enhanced by using electron transfer dissociation (ETD) for the analysis of large endoproteinase Lys-C products.22 However, it is not mandatory to use endoproteinase Lys-C as part of the IPTL strategy. Tryptic digests would be just as appropriate for IPTL analysis using the chemicals described in this report, provided that the data interpretation software is taking into account that only peptides with a C-terminal lysine will produce data that are suitable for quantification. Alternatively, complete isobaric labeling of tryptic digests could be achieved using a three-step modification strategy. This strategy could consist of, for example, modification of C-terminal lysines with MDHI, followed by N-terminal modification of the peptides with succinic anhydride (12C4 and 13C4), and proteolytic incorporation of two oxygens using oxygen-18 versus oxygen16 at the C-terminus.5 The availability of IPTL-specific data interpretation tools will significantly increase the usefulness of this novel approach to relative protein quantification. Other future developments of the method include the modification of the IPTL reagents to allow for the multiplexed analysis of three or more samples. Similar to the use of known amounts of iTRAQ labeled peptides as internal standards for absolute protein quantification,23 IPTL-labeled peptides can also be used as a straightforward and affordable tool for absolute protein quantification.

Conclusion Here, we present isobaric peptide termini labeling, a novel approach to isobaric protein quantification that retains all the known advantages of isobaric labeling strategies. IPTL has significant advantages for protein quantification using mass spectrometers with limited capabilities in the low molecular mass range and the presence of several quantification points in each MS/MS spectrum provides the ability to perform statistical data analysis and increased quantitative robustness. The data presented here have been analyzed using a conceptually very simple quantification algorithm that is based on calculating Mascot score ratio values. A more detailed approach to IPTL data analysis is in progress and will be reported in due course. Abbreviations: CID, collision induced dissociation; ETD, electron transfer dissociation; GIST, global internal standard technology; ICAT, isotope-coded affinity tagging; ICPL. isotope4340

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coded protein labeling; IPTL, isobaric peptide termini labeling; iTRAQ, isobaric tagging for relative and absolute quantification; MDHI, methoxy-4,5-dihydro-1H-imidazole; SA, succinic anhydride; SILAC, stable isotope labeling by amino acids in cell culture; STLC, S-Trityl-L-cysteine; TMT, tandem mass tag. Declaration of Conflict of Interest. A.T. is working for NEPAF, a contract proteome analysis facility that is offering IPTL-based protein quantification as a service.

Acknowledgment. The present study was supported by the National Program for Research in Functional Genomics in Norway (FUGE) of the Norwegian Research Council. Supporting Information Available: List of all identified proteins after incubation of HeLa cells with STLC for 12 and 24 h using IPTL, including Mascot scores, number of matched queries, sequence coverage, number of unique peptides, and relative protein scores. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Ong, S. E.; Mann, M. Mass spectrometry-based proteomics turns quantitative. Nat. Chem. Biol. 2005, 1 (5), 252–262. (2) Bantscheff, M.; Schirle, M.; Sweetman, G.; Rick, J.; Kuster, B. Quantitative mass spectrometry in proteomics: a critical review. Anal. Bioanal. Chem. 2007, 389 (4), 1017–1031. (3) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 1999, 17 (10), 994–999. (4) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 2002, 1 (5), 376–386. (5) Miyagi, M.; Rao, K. C. Proteolytic 18O-labeling strategies for quantitative proteomics. Mass Spectrom. Rev. 2007, 26 (1), 121– 136. (6) Panchaud, A.; Affolter, M.; Moreillon, P.; Kussmann, M. Experimental and computational approaches to quantitative proteomics: status quo and outlook. J. Proteomics 2008, 71 (1), 19–33. (7) Chakraborty, A.; Regnier, F. E. Global internal standard technology for comparative proteomics. J. Chromatogr., A 2002, 949 (1-2), 173–184. (8) Schmidt, A.; Kellermann, J.; Lottspeich, F. A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics 2005, 5 (1), 4–15. (9) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.;

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