Combining low- and high-energy tandem mass spectra for optimized peptide quantification with isobaric tags

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1 available at Combining low- and high-energy tandem mass spectra for optimized peptide quantification with isobaric tags Loïc Dayon a, Carla Pasquarello b, Christine Hoogland c, Jean-Charles Sanchez a,b, Alexander Scherl b, a Biomedical Proteomics Group, Department of Structural Biology and Bioinformatics, Faculty of Medicine, University of Geneva, Geneva, Switzerland b Proteomics Core Facility, Faculty of Medicine, University of Geneva, Geneva, Switzerland c Proteome Informatics Group, Swiss Institute of Bioinformatics, Geneva, Switzerland ARTICLE INFO Article history: Received 31 August 2009 Accepted 31 October 2009 Keywords: Quantitation ESI itraq Mass spectrometry Orbitrap Proteomics Tandem mass tags TMT ABSTRACT Isobaric tagging, via TMT or itraq, is widely used in quantitative proteomics. To date, tandem mass spectrometric analysis of isobarically-labeled peptides with hybrid ion trap orbitrap (LTQ-OT) instruments has been mainly carried out with higher-energy C-trap dissociation (HCD) or pulsed q dissociation (PQD). HCD provides good fragmentation of the reporter-ions, but peptide sequence-ion recovery is generally poor compared to collisioninduced dissociation (CID). Herein, we describe an approach where CID and HCD spectra are combined. The approach ensures efficiently both identification and relative quantification of proteins. Tandem mass tags (TMTs) were used to label digests of human plasma and LC- MS/MS was performed with an LTQ-OT instrument. Different HCD collision energies were tested. The benefits to use CID and HCD with respect to HCD alone were demonstrated in terms of number of identifications, subsequent number of quantifiable proteins, and quantification accuracy. A program was developed to merge the peptide sequence-ion m/z range from CID spectra and the reporter-ion m/z range from HCD spectra, and alternatively to separate both spectral data into different files. As parallel CID in the LTQ almost doesn't affect the analysis duty cycle, the procedure should become a standard for quantitative analyses of proteins with isobaric tagging using LTQ-OT instruments Elsevier B.V. All rights reserved. 1. Introduction Isotopic dilution combined with mass spectrometry (MS) has become the method of choice for discovery-based proteomics. Different strategies are used to introduce an isotopic label into a complex biological sample. Typically, the label can be introduced during cell growth [1 3] or during the sample preparation at the protein level (as, for example, ICAT [4]) or at the peptide level. Labeling peptides after proteolytic digestion is mainly performed with isobaric tags such as tandem mass tags (TMTs) or itraq [5 7]. These isobaric compounds are synthesized with heavy and light isotopes to have the same total mass, but to give rise to reporter-ions at different masses after activation and subsequent tandem mass spectrometry (MS/MS or MS2). Activation is generally done using collision-induced dissociation (CID). Relative quantification between protein content of Corresponding author. Proteomics Core Facility, Centre Medical Universitaire, Rue Michel-Servet 1, 1211 Genève 4, Switzerland. Tel.: ; fax: address: Alexander.Scherl@unige.ch (A. Scherl) /$ see front matter 2009 Elsevier B.V. All rights reserved. doi: /j.jprot

2 770 JOURNAL OF PROTEOMICS 73 (2010) different samples is then performed according to the ratio of the reporter-ion abundances in the MS2 spectrum. Isobaric tags have two major advantages compared to quantification in the first stage of mass spectrometry (MS1). First, this mode of operation provides an increased signal to noise ratio of reporter-ions used for quantification due to the removal of chemical noise in the second stage of mass spectrometry. Second, all differentially labeled precursor ions have the same mass. Thus, the MS1 spectrum is less complex, improving data-dependent precursor ion selection for indepth characterization of the sample. On the other hand, relatively high collision energy has to be applied in order to obtain good reporter-ion statistics. However, higher collision energy is not suitable to obtain peptidic backbone ions used to match experimental MS2 spectra to peptides from protein sequence databases. In addition, the relatively low reporterion masses (114 to 117 Da for itraq and 126 to 131 for TMT) are not suited for ion trap instruments. Indeed, these lowmass ions are not stable during the activation step, a principle known as the 1/3rd rule [8]. Recently, hybrid instruments combining the high sensitivity and versatility of linear ion traps with high performance orbitrap (OT) analyzers became available [9,10]. They have rapidly become popular for proteomics experiments. Basically, two activation methods using CID can be used with hybrid OT instruments for the analysis of low-mass fragment ions such as isobaric tag reporter-ions. The so-called pulsed q dissociation or PQD [11,12] consists of activating the precursor ion for a very short time at a high q value, where q is the Mathieu parameter, proportional to the applied radiofrequency voltage in the ion trap. The q value is then lowered before the dissociation of the precursor ion, allowing low-mass ions to be trapped as well. However, the sensitivity of this mode of operation is low compared to traditional CID [13]. Also, the relatively low resolution of the ion trap does not allow precise quantification on closely separated ions such as isobaric tag reporter-ions. Higher-energy C-trap dissociation (HCD) is the second activation method for the analysis of low-mass fragment ions. Activation and dissociation take place between the C-trap, used for the ion storage between the linear ion trap and the OT analyzer, and a supplementary hexapole placed after the C-trap [14]. This fragmentation mode produces higher collision energy that efficiently generates the reporter fragments for quantification. However, this higher collision energy is associated with a decreasing sensitivity due to ion scattering and a lack of backbone fragments useful for peptide identification [15]. Optimized quantitative analysis of phosphopeptides with itraq labeling has been recently reported and it has been shown that classical CID yielded better phosphopeptide sequence identification compared to HCD [16]. The authors proposed to combine both modes for the quantitative analysis of phosphopeptides with itraq. In the same way, this strategy has been applied very recently to study the changes in protein expression in mouse hearts upon transverse aortic constriction with CID followed by HCD of the precursor ions [17]. Better performance with a CID/HCD hybrid acquisition strategy was demonstrated relative to PQD acquisition. The authors demonstrated the superior sensitivity and limit of quantification on digested bovine serum albumin (BSA) and applied their quantification strategy to a complex protein mixture. In this report, we demonstrate that combining CID and HCD activation modes in a linear ion trap orbitrap (LTQ-OT) hybrid instrument allows precise peptide quantification without compromising peptide identification with the linear ion trap analyzer. To do so, a higher-energy HCD spectrum and a low energy CID spectrum were acquired on each selected precursor ion. The CID spectrum was then used for peptide identification at highest sensitivity, and the HCD spectrum was used for precise peptide quantification. TMTs were used as isobaric labeling reagents. Different HCD collision energies were evaluated and their impact on peptide/protein identification and quantification was carefully assessed. A program, accessible over the internet, and compatible with all database search tools for the creation of composite spectra containing both qualitative and quantitative information was developed. Because this program can also dissociate CID and HCD spectra, it can be used for different data analysis schemes, as for example a separated analysis pipeline for CID and HCD spectra. Finally, we showed how this combined acquisition mode affected the instrument duty cycle and the number of quantifiable peptides. 2. Experimental section 2.1. Materials Human plasma, iodoacetamide (IAA, 99%) and tris(2- carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma (St. Louis, MO, USA). Triethylammonium hydrogen carbonate buffer (TEAB) 1 M ph=8.5 and sodium dodecyl sulphate (SDS, 98%) were from Fluka (Buchs, Switzerland). Formic acid (FA, 99%) was from Biosolve (Valkenswaard, the Netherlands). Hydroxylamine solution 50 wt.% in H 2 O(99.999%) was from Aldrich (Milwaukee, WI, USA). Hydrochloric acid (25%) was from Merck (Darmstadt, Germany). Water for chromatography LiChrosolv and acetonitrile Chromasolv for HPLC ( 99.9%) were respectively from Merck and Sigma- Aldrich (Büchs, Switzerland). The duplex Tandem Mass Tags (TMT ) were kindly provided by Proteome Sciences (Frankfurt am Main, Germany) and can be purchased from Thermo Scientific (Rockford, IL, USA). Sequencing grade modified trypsin was from Promega (Madison, WI, USA) Sample preparation Ten µl of plasma was dissolved in 990 µl of TEAB 100 mm adjusted to ph=8 with diluted HCl. To 200 µl of this solution, 2 µl of SDS 1% and 4 µl TCEP 50 mm were added. The reduction was carried out at 60 C for 1 h. Alkylation was performed by adding 2 µl of IAA 400 mm during 30 min in the dark. Twenty µl trypsin 0.2 µg µl 1 (freshly prepared in the above-mentioned TEAB solution) was added and the digestion was proceeded overnight at 37 C. The sample was divided into two identical aliquots. Duplex TMT labeling was achieved for 1 h, after addition of 40.3 µl of TMT duplex reagent in CH 3 CN (i.e., 0.83 mg, mol). One plasma aliquot was labeled with TMT with reporter-ion at m/z=126.1 and the other with TMT with reporter-ion at m/z= Then, 8 µl of hydroxylamine 5% was added in each tube for reaction during 15 min. The differentially TMT-labeled samples were pooled

3 771 in a new tube and the resulting mixture was dried. The sample was desalted with Oasis HLB 1cm 3 (30 mg) extraction cartridges from Waters (Milford, MA, USA) following the manufacturer's instructions. After drying, the sample was cleaned again with a C18 ultramicrospin column (Harvard Apparatus, Holliston, MA, USA), and dried. The sample was dissolved in H 2 O/CH 3 CN/FA 96.9/3/0.1 for MS analysis LC-MS/MS LC-MS/MS was performed on a LTQ orbitrap XL from Thermo Electron (San Jose, CA, USA) equipped with a NanoAcquity HPLC system from Waters. Tryptic peptides (either 2 µg or 0.1 µg) were trapped on a home-made 5 µm 200 Å Magic C18 AQ (Michrom) mm pre-column. Following washing, they were separated on a home-made 5 µm 100 Å Magic C18 AQ (Michrom) mm column with a gravity-pulled emitter. The analytical separation was run for 85 min using a gradient of H 2 O/FA 99.9/0.1 (solvent A) and CH 3 CN/FA 99.9/0.1 (solvent B). The gradient was run as follows: 0 1 min 95% A and 5% B, then to 65% A and 35% B at 55 min, and 20% A and 80% B at 65 min at a flow rate of 220 nl min 1, followed by re-equilibration of the column. For MS1 survey scans, the OT resolution was 60,000 and the ion population with an m/z window from 400 to For MS/MS in the LTQ, the ion population was (isolation width of 2 m/z units). For MS/MS detection in the OT with HCD activation, ion population was set to (isolation width of 4 m/z units), with a resolution of 7500, first mass at m/z=100, and a maximum injection time of 750 ms. A maximum of three precursor ions (most intense) were selected for activation and subsequent MS/MS analysis. CID was performed at 35% of the normalized collision energy (NCE) in all cases. Different collision energies were evaluated for HCD, i.e., 30%, 40%, 50%, 60% and 70% NCE. Control experiments were carried out with CID alone. In this case, the five most intense precursor ions were selected to maximize peptide/ protein identification. The different operating modes are detailed precisely in the Results and discussion part. Briefly, one acquisition mode used HCD alone as fragmentation method and the other performed parallel fragmentation of a given precursor by CID and HCD (CID/HCD mode). All analyses were performed in triplicates. The resulting.mgf files were searched against UniProt- Swiss-Prot database (57.4 of 16-Jun-2009) using Mascot (version 2.2, Matrix Sciences, London, UK) and Phenyx (version 2.6, GeneBio, Geneva, Switzerland). Homo sapiens taxonomy was specified for database searching. Variable amino acid modifications were oxidized methionine. TMT-labeled peptide amino terminus and TMT-labeled lysine ( Da) were set as fixed modifications as well as carbamidomethylation of cysteines. Trypsin was selected as the enzyme, with one potential missed cleavage. As to Mascot, peptide and fragment ion tolerance was respectively 20 ppm and either 0.6 or 0.02 Da when fragments were recorded in the LTQ or in the OT. The significance threshold (p) was set to The Mascot ion score cutoff value was then automatically derived to 30 by the software. In Phenyx, turbo scoring was selected. Falsepositive ratios were estimated using a reverse decoy database [18]. All datasets where searched once in the forward and once in the reverse database. Separate searches were used to keep the database size constant. Protein and peptide score were then set up to maintain the false-positive peptide ratio below 1%. This resulted in a slight overestimation of the false-positive ratio [18]. Parent ion tolerance was set to 20 ppm. The scoring model was selected depending on fragment analysis, either in the LTQ or in the OT. For LTQ scans, the peptide z-score cutoff was set to 5.2. For HCD scans, the peptide z-score cutoffs were set to 9.9, 8.6, 8.2, 7.3, and 7.3 for respective NCE of 30%, 40%, 50%, 60%, and 70% Protein and peptide quantification Reporter-ion abundances (I 126 and I 127 ) were extracted directly from peak lists using dedicated Phenyx exports. No individual protein quantification was carried out. I 126 and I 127 were corrected from isotopic impurities [7,19]. Relative quantification ratios were calculated by division of the corrected abundances of reporter-ions at m/z=127.1 (i 127 ) over those of reporter-ions at m/z=126.1 (i 126 ). Average ratio and mean relative standard deviation (RSD) were calculated on the normalized data i 126 / (i 126 +i 127 )andi 127 /(i 126 +i 127 ) according to the Libra module used in the trans-proteomic pipeline (TPP). In addition, geometric average ratio and RSD were also calculated on the ratios i 126 /i 127 according to the Mascot quantification module Protein and peptide identification Peak lists files (.dta) were generated using the extract_msn. exe embedded in the data analysis software provided by the instrument vendor. Then, the.dta files were merged into a.mgf( mascot generic format ) file using an in-house perl script. CID and HCD data were merged using an in-house perl program as well. This script basically kept, for a given precursor ion, the CID fragments (m/z and ion abundance), and added to their mass list the HCD fragments (m/z and ion abundance) comprised in a specified m/z window (typically in the zone of reporter-ions). An m/z window between 124 and 129 was specified using the duplex TMTs. An additional function of this software was to separate peak lists whose fragments were originally acquired in the LTQ or in the OT in order to extract data coming from HCD fragmentation only. 3. Results and discussion In order to optimize both peptide/protein identification and quantification when analyzing isobarically-tagged peptides with LTQ-OT MS, two MS/MS acquisition modes were evaluated. One was carried out using HCD alone with analysis of the fragment ions in the OT. Different collision energies were assessed. The other mode was performed with concomitant fragmentation by CID and HCD (CID/HCD mode) with fragment ion analysis respectively in the LTQ and OT. In both cases, all MS1 survey scans were acquired in the OT analyzer. A human plasma sample was taken as test sample. After reduction, alkylation and digestion with trypsin, identical plasma samples were labeled with different versions of the duplex TMT and mixed. The pooled sample was analyzed with RP-LC LTQ- OT MS.

4 772 JOURNAL OF PROTEOMICS 73 (2010) CID vs. HCD regarding peptide and protein identification Peptide and protein identification from MS2 spectra depends mainly on mass measurement accuracy of precursor and fragment ions, peptide sequence coverage by fragment ion series and the algorithm and parameters used for the database search. The identification performance of TMT-labeled peptides was evaluated according to the experimental set-up. HCD fragmentation was performed with increasing collision energies ranging from 30% to 70% NCE. The number of unique peptides identified with Mascot was reported as a function of the collision energy in Fig. 1a. This number increased to reach a plateau for energies of 40% and 50% NCE and then it decreased drastically. At 70% NCE, only 8.9% of unique peptides were identified with respect to the maximum number at 50% NCE (i.e., 719 unique peptides). An identical behavior was obtained with 20-times less sample to analyze (i.e., 0.1 µg). The results were slightly different in comparison to a very recent report [17] where collision energies of 30% and 40% were optimal for the analysis of itraq-labeled tryptic peptides of BSA. The data obtained by acquiring CID and HCD spectra together (CID/HCD mode) were merged using a program written in-house. The program added the HCD spectrum window containing the reporter-ion m/z region into the corresponding CID spectrum for each precursor ion. Ion abundance was then normalized so that the base peak of the CID spectrum corresponded to the base peak of the reporter-ion region of the HCD spectrum. Using this procedure the number of identified unique peptides was slightly higher (respectively 745 and 763 unique peptides for HCD collision energy of 50% and 60%) compared to HCD alone at 40% and 50% collision energy (Fig. 1a). Because identifications were obtained from CID data, this number was reproducible whatever HCD collision energy was used. A control comparison with the gold standard method for peptide identification (i.e., CID alone, see Experimental section) stressed that the quantification procedure presented herein reduced the total number of potential identifications as a consequence of the increased scanning duty cycle [15]. With the database search engine Phenyx, HCD spectra acquired at 40% and 50% NCE provided slightly more identifications than those obtained by CID/HCD (whatever HCD collision energy was used; see Supplementary information SI1). Indeed, the optimized Phenyx algorithm was previously shown to take good advantage of the high measured mass accuracy of fragment ions [15]. The total number of matched peptides was also evaluated for both HCD and CID/HCD approaches (Fig. 1b). This number was actually relevant as all these peptides served for protein quantification. In general, the higher number of matched peptides per protein increases the value of the quantitative measurements in terms of accuracy, precision, and overall Fig. 1 Number of unique peptides (a) and total number of peptides (b) identified with Mascot according to the acquisition mode used for the RP-LC LTQ-OT MS analysis. Several collision energies were tested for HCD, ranging from 30% to 70%. Either 2 µg (light bars) or 0.1 µg (dark bars) of TMT-labeled human plasma digest were loaded on LC column. Control CID experiments were carried out and were reported in the upper figure. Fig. 2 Venn diagram of unique peptides identified with Mascot when performing the search on the merged CID/HCD data (i.e., identification carried out on CID spectra) or on the dissociated (i.e., extracted) HCD spectra. Using the CID/HCD acquisition mode with the LTQ-OT mass spectrometer, collision energies of 50% (a) and 60% (b) for HCD were evaluated. Two µg of TMT-labeled human plasma digest was loaded on LC column. The calculation was performed from a common database search of the triplicate MS analyses.

5 773 statistics. A 10% increase of matched peptides was obtained using the CID/HCD mode with respect to HCD at 50% collision energy. The concomitant fragmentation of peptides using the CID/ HCD mode with respective analysis in the LTQ and OT analyzer allowed performing two independent database searches and subsequent relative quantification (i.e., on the merged CID/HCD spectra, and on the extracted HCD spectra). After extracting the HCD peak list from the whole dataset with our program, and performing the database search, different peptides were identified. This is illustrated in the Venn diagram of Fig. 2. Comparison of Fig. 2a and b obtained at respectively 50% and 60% HCD collision energy showed again that HCD performed at 60% was detrimental to the identification process when CID mass spectra were not acquired in parallel. Proportionally more peptide identifications were obtained from the extracted HCD spectra with Phenyx (see Supplementary information SI2). At 50% collision energy, 94 and 98 unique peptides were respectively identified only using the merged CID/HCD and the extracted HCD data. Ultimately, successive database searches with different search engines are therefore a relevant option to increase the total number of peptide/protein identification. The situation where a different peptide sequence was identified from the two CID and HCD scans did not occur in this dataset. Also, the number of accidental CID events [20] (e.g., MS2 spectra with more than one peptide sequence identified after co-isolation and cofragmentation) was low in both CID and HCD spectra (below 10), although a wider isolation window was used for the HCD spectra. In practice, a wider isolation window is necessary to increase the sensitivity of the HCD mode with OT acquisition to a similar level as the CID mode with linear ion trap acquisition [15] Influence of collision energy on the quantification Regarding peptide identification, HCD and CID/HCD could provide equivalent results. Quantification results enabled to refine the selection of an accurate acquisition mode for the analysis of samples modified with isobaric tags such as TMT. Quantification performances were first assessed using HCD alone (collision energy ranging from 30% to 70% NCE). From 30% to 50%, the best quantitative results were obtained at collision energy of 50% in terms of reporter-ion abundance, ratio accuracy and precision (Table 1 and Fig. 3a). At this energy value, the relative standard deviation (RSD) calculated on the relative abundance according to the Libra module used in the trans-proteomic pipeline (TPP) was 3.8%. The RSD value at 30% collision energy was 26.0%, rendering precise quantification impossible (median and average ratios were 1.02, and 1.01 respectively). Although 40% NCE displayed efficient identification results (Fig. 1), the RSD was 17.7%. The results for collision energy of 50%, 60% and 70% are reported in Fig. 3b. Although less data points were obtained when performing HCD alone at 60% and 70% collision energy (as less peptide matches were obtained; see Fig. 1 and Supplementary information SI1), the precision of the relative quantification was superior with respective RSD values of 3.0% and 2.9%. As shown in Table 1, average ratios calculated according to the Libra or Mascot quantification module were identical. The associated RSD and geometric RSD followed similar tendencies. We also investigated if absolute reporter-ion abundance influenced the quantification accuracy. Table 2 shows the Table 1 Summary of the performances of HCD and CID/HCD acquisition modes tested on a human plasma tryptic digest sample labeled with duplex TMT and analyzed with RP-LC LTQ-OT MS (2 µg on column). Data were obtained from the triplicate MS analyses. Number of MS2 spectra d Missing values c /% Ratio geometric RSD b /% Geometric average ratio b ð i127=i126þ Relative abundance mean RSD a /% Abbreviation Median ratio Average ratio a ð i127=ði126 + i127þ= i 126=ði126 + i127þþ Collision energy for HCD/% NCE MS/MS acquisition mode HCD 30 HCD ±85 40 HCD ±55 50 HCD ±76 60 HCD ±77 70 HCD ±7 CID/HCD 50 CID/HCD ±37 60 CID/HCD ±35 Average ratio and mean RSD values were calculated on the relative abundance i126/(i126+i127) and i127/(i126+i127) according to the Libra module used in the trans-proteomic pipeline (TPP). Geometric average ratio and RSD values were calculated on the ratios i127/i126 according to the Mascot quantification module. Missing values corresponded to the percentage of reporter-ion without reporter signal over the total number of reporter-ions. d For CID/HCD, the number of MS2 spectra corresponded to the number of MS2 spectra after combination of CID and HCD spectra together. During acquisition, the actual number of MS/MS events was therefore twice more. a b c

6 774 JOURNAL OF PROTEOMICS 73 (2010) Fig. 3 Distribution representation of Log2(i 127 /i 126 ) as a function of the HCD collision energy. The MS/MS experiments were carried out with HCD alone. The collision energy was varied between 30 and 50% (a), and 50 and 70% (b). Two µg of TMT-labeled human plasma digest was loaded on LC column. Data were obtained from the triplicate MS analyses.

7 775 Table 2 Quantification performances of CID/HCD acquisition mode tested on a human plasma tryptic digest sample labeled with duplex TMT and analyzed with RP-LC LTQ-OT MS (2 µg on column). Data were obtained from the triplicate MS analyses. Experiment Reporter intensity/counts Number of peptides a Average ratio b ð i 127 =ði i 127 Þ= i126 =ði i 127 ÞÞ Relative abundance mean RSD b /% Geometric average ratio c ð i 127 =i 126 Þ Ratio geometric RSD c /% CID/HCD CID/HCD a The number of peptides corresponded to the sum of the peptides from the triplicate MS analyses. b Average ratio and mean RSD values were calculated on the relative abundance i 126 /(i 126 +i 127 ) and i 127 /(i 126 +i 127 ) according to the Libra module used in the trans-proteomic pipeline (TPP). c Geometric average ratio and RSD values were calculated on the ratios i 127 /i 126 according to the Mascot quantification module. measured average peptide ratio and RSD as a function of the absolute reporter-ion count. Basically, the accuracy of the measured ratio and the RSD respectively increased and decreased when the intensity of the reporter-ions increased. Reporter-ions with abundances between and counts provided excellent accuracy and RSD. Collision energy of 60% NCE provided more reporter-ions in this range of intensity. Above counts, the few numbers of data did not allow to evaluate the accuracy and potential saturation issues were observed. These results showed that a high absolute ion count was beneficial for precise quantification. Furthermore, sufficient collision energy prevented obtaining missing values (zero values), reflecting the lack of detection of TMT reporter-ions within the OT analyzer (Table 1). With a lower amount of sample to analyze (i.e., 0.1 µg), the quantification was also better when higher energy was used (see Supplementary information SI4 and SI5).Inthisreport,anionisolationwindowof4m/z units for HCD spectra was chosen. As discussed previously, a larger isolation window increases the number of ions ultimately detected in the Fourier-transform analyzer (in this case the OT). It also reduces the duty cycle by decreasing the ion injection times, and increases the ion statistics [15], and thus the quantification accuracy. On the other hand, opening the precursor ion isolation window might increase the number of accidental CID events and thus perturb the quantification accuracy. Sample fractionation prior to LC-MS/MS analysis should be considered in this case to reduce the number of accidental CID events and thus provide best quantification accuracy. As shown here, HCD energy had to be properly tailored in order to obtain valuable quantitative analysis. When using HCD alone, collision energy of 50% NCE was absolutely required to ensure both valuable identification and quantification Enhanced identification and quantification by merging CID and HCD spectra With the combined CID/HCD acquisition mode, peptide identification through CID spectra could be ensured whatever collision energy was used for HCD. Collision energy of 60% NCE could be implemented without being detrimental to the identification process (Fig. 4a). Besides, quantification performances were enhanced as well. Comparison of Figs. 3b and 4a clearly highlighted the combined benefit in peptide identification rate and quantification accuracy. Higher collision energy provided higher reporter-ion signals (Fig. 4b), preventing acquisition of missing values (Table 1) and potentially allowing higher limits of quantification (see Table 2 and Supplementary information SI3). The combined CID/HCD acquisition mode at 50% NCE offered a good compromise for proteomics discovery experiments. Quantitative data were sufficiently accurate and precise (RSD of 4.5%). Identification could be carried out sequentially on merged CID/HCD and extracted HCD spectra (Fig. 2a and Supplementary information SI2ba), both allowing accurate relative quantification. When accuracy, precision and/or limit of quantification become an issue, higher collision energy should be used for HCD in order to favor the release of TMT reporter-ions. This is especially relevant when targeted proteomics approaches are involved. The combined CID/HCD mode did almost not affect the instrument duty cycle. The parallel acquisition mode (e.g., CID scans were performed in the linear ion trap while the transient signal was recorded in the OT analyzer) allowed acquiring the 3 CID spectra during the MS1 survey scan. Average spectrum acquisition time for an MS1 survey scan was 1.06 s. Average scan time for an MS2 spectrum acquisition in the linear ion trap was 0.27 s. The acquisition of the 3 CID scans in the linear ion trap was therefore performed on the same timescale as the MS1 survey scan. In contrast, the average scan time for a HCD spectrum acquisition was 0.88 s. The 3 HCD acquisitions were performed after the MS1 survey scan. These HCD scans, necessary for quantification with isobaric tags, were therefore responsible for the largest loss in the duty cycle. The total number of selected precursor ions for each acquisition mode is shown in Table 1. In summary, the 3 added CID scans in the linear ion trap were coming almost for free with the

8 776 JOURNAL OF PROTEOMICS 73 (2010) Fig. 4 Distribution representation of Log2(i 127 /i 126 ) as a function of the HCD collision energy (a). The MS/MS experiments were carried out with the CID/HCD acquisition mode. Identification was obtained from CID fragments and quantification from the TMT reporter-ions under HCD. HCD collision energies of 50% and 60% were tested. Abundance of reporter-ions at m/z=127.1 (i 127 ) was represented as a function of reporter-ion abundance at m/z=126.1 (i 126 ) (b). A log scale was used. Two µg of TMT-labeled human plasma digest was loaded on LC column. Data were obtained from the triplicate MS analyses.

9 777 combined method presented here. In average, the number of fragmented precursor ions decreased by less than 20% and this number was easily compensated by the superior sensitivity of the CID spectrum used for peptide identification. 4. Conclusions Concomitant CID and HCD MS/MS acquisition mode was demonstrated to be valuable in terms of identification and quantification when analyzing TMT-labeled peptides with a hybrid linear ion trap orbitrap mass spectrometer. Because CID and HCD can be carried out in parallel respectively with analysis in the LTQ and in the OT, the CID/HCD operating mode almost doesn't affect the overall scanning duty cycle. There is therefore no disadvantage to use it instead of performing HCD alone. The appropriate HCD collision energies for accurate quantification have been emphasized. Informatics tools were developed to deal with the data obtained with the combined CID/HCD mode. One is dedicated to the merging of the CID spectra with corresponding reporter-ion signals from the HCD spectra. The other is used to separate peak lists from CID and HCD spectra in order to proceed to a second identification and quantification round on the extracted HCD data. These programs are available on the expasy website ( In conclusion, the described procedure should rationally become a standard for the quantitative analyses of proteins with isobaric tagging using LTQ-OT mass spectrometers. Acknowledgment The authors thank Proteome Sciences R&D GmbH & Co. KG for the financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: /j.jprot REFERENCES [1] Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. 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