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A. Voráˇc et al., Eur. J. Mass Spectrom. 20, 255–260 (2014) Received: 19 October 2013 n Revised: 19 February 2014 n Accepted: 7 April 2014 n Publication: 22 April 2014
EUROPEAN JOURNAL OF MASS SPECTROMETRY
A simplified method for peptide de novo sequencing using 18O labeling Aleš Voráˇc,a Ondrej Šedo,a,b Jan Havliša,b and Zbynˇ ek Zdráhala,b a Research Group Proteomics, Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic. E-mail:
[email protected] b
National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
Incorporation of an 18O atom into a peptide C-terminus by proteolytic cleavage in the presence of H218O is one of the most effective ways of enhancing tandem mass spectrometry (MS/MS)-based de novo sequencing. Incorporation is usually accomplished by procedures including vacuum-assisted drying of tryptic peptides extracted from gels, their subsequent reconstitution in a H216O/H218O mixture and re-treatment with trypsin. In the present work, we propose a simplified procedure for 18O incorporation into tryptic peptides by adding H218O and trypsin to the original digest solution. In comparison to published methods, the proposed protocol for peptide de novo sequencing brings significant advantages in analysis and workflow with no deterioration in method performance. We show that labeling by this simplified method leads to a highlighting of the y-ion fragment series in the peptide matrix-assisted laser desorption/ionization (MALDI)-MS/MS data, which facilitates MS/MS data interpretation. We also prove that eliminating acid extraction of peptides from gels does not result in a decrease in sequence coverage or a qualitative loss of particular peptides detectable by MALDI-MS. The method was examined by MALDI-MS/MS on bovine serum albumin and recombinant histidine kinase CKI1 from Arabidopsis thaliana, and was verified by de novo sequencing of tryptic peptides originating from Apodemus sylvaticus salivary proteins. Keywords: peptide de novo sequencing, mass spectrometry, isotopic labeling, 18O incorporation
Introduction De novo sequencing of peptides is essential in the analysis of proteins from organisms with unsequenced genomes, protein isoforms, proteins with modified amino acids, etc. For this purpose, techniques based on tandem mass spectrometry (MS/MS) are currently the most often employed tools. However, interpretation of peptide MS/MS spectra is often complicated due to the presence of internal fragment ions. This leads to uncertainty in the assignment of particular MS/MS fragments to an appropriate ion series, delaying a correct peptide-sequence identification. Possible ways of enhancing peptide de novo sequencing by MS lie in distinguishing C-terminal (e.g. y-series) and N-terminal (e.g. b-series) fragment ions in the MS/MS data by modification of the C- or N-terminus of the peptide, which leads to a specific mass shift in its C-terminal or N-terminal fragment ions as compared to the unmodified peptide. ISSN: 1469-0667 doi: 10.1255/ejms.1277
For reliable and easy MS/MS spectral interpretation, the incorporation of 18O atoms into the carboxyl group of the peptide C-terminus was reported.1–2 MS/MS analysis of 18O-labeled peptides showed a characteristic isotopic distribution of their C-terminal fragment ions in contrast to the normal isotopic distribution of their N-terminal fragment, internal fragment and iminium ions. An 18O atom can be incorporated into the peptide C-terminus by acid-catalyzed exchange,3 esterasecatalyzed exchange,4 or, more reliably, by protease-catalyzed hydrolysis of the peptide bonds in the presence of H218O.5 One 18 O atom is directly incorporated into the C-terminal carboxyl group of each proteolytically generated peptide by protease hydrolysis. Subsequently, proteases can exchange a second 16 O atom of the carboxyl group with 18O.6 It was found that proteases Lys-N and Asp-N tend to incorporate one 18O atom © IM Publications LLP 2014 All rights reserved
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Simplified Method for Peptide de novo Sequencing using 18O Labeling
and proteases Lys-C and trypsin tend to incorporate two 18O atoms in total.5,6 In contrast to chemical labeling, no side reactions occur in the peptide chain.7 Additionally, proteases are routinely used for protein digestion in “bottom-up” proteomics strategies. The combination of cleavage and labeling in one step does not affect the efficiency of the enzyme and eliminates the necessity of incorporating an additional chemical derivatization step into the analytical workflow, a source of possible errors. Owing to its high specificity and efficiency, trypsin is the most commonly used protease for cleavage and labeling. Furthermore, tryptic peptides tend to provide long continuous y-series ions, as a basic residue is located at their C-terminus.8 Up to now, several variants of the 18O labeling procedure have been developed. For subsequent de novo sequencing, an 18O atom is typically incorporated into the peptide C-terminus by proteases in a buffer containing 50% H216O and 50% H218O (v/v).2,9–11 Shevchenko et al. demonstrated that proteins can also be labeled by 18O during in-gel digestion.9,11 Schnolzer et al.5 recommended division of the protein sample into two halves: one half to be cleaved in the presence of H216O and the other one in the presence of H218O. After labeling, the two halves were mixed in a 1 : 1 ratio and analyzed by MS.5 Kuster and co-workers divided protease cleavage and 18O-labeling into two separate steps.7 In the first step, the tryptic cleavage of proteins in the gel was accomplished, while in the second step the resulting peptides were vacuum-dried and re-dissolved in a buffer containing trypsin and H218O. Hajkova et al. also recommended this method because of the different optimum conditions for each reaction.12 Routinely, peptides are obtained from gels by acid extraction. The immediate action of trypsin in the resulting solution is impossible and, therefore, vacuum-assisted drying needs to be employed prior to the labeling reaction. However, this step is accompanied by possible materialloss.13–15 In the present paper, we describe a simplified method based on the direct labeling of peptides by the addition of H218O and trypsin to the solution produced by routine proteolysis in H216O. The output of the method is compared with the published two-step procedure7 and with another, principally different, labelingapproach utilizing reductive dimethylation.16
Materials and methods Chemicals and reagents
Histidine kinase CKI1 from Arabidopsis thaliana, expressed in E. coli, was obtained from a collaborating group (Functional Genomics and Proteomics of Plants, Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology). Two-dimensional polyacrylamide gel electrophoresis (PAGE) spots containing proteins isolated from Apodemus sylvaticus saliva (acquired from the Department of Zoology, Faculty of Science, Charles University) were prepared using a procedure described elsewhere.16 Bovine serum albumin (BSA), ammonium bicarbonate (AB), formic
acid (FA), formaldehyde, sodium acetate, sodium cyanoborohydride, acetic acid, ammonium hydroxide and trifluoroacetic acid (TFA) were purchased from Sigma Aldrich (St Louis, Missouri). Isotopically enriched water H218O (97% pure) was obtained from Cambridge Isotope Laboratories, Inc. (Andover, Massachusetts). Acetonitrile was purchased from Merck (Darmstadt, Germany). Sequencing grade trypsin was obtained from Promega (Madison, Wisconsin). Alpha-cyano4-hydroxycinnamic acid (CHCA) was purchased from Bruker Daltonics (Bremen, Germany). All solutions were prepared using water purified by a Milli-Q plus 185 systems (Millipore, Billerica, Massachusetts).
Sample treatment Three parallel samples of sodium dodecyl sulfate PAGE bands of CKI1 (5 µg of protein was loaded per gel lane), BSA (12, 6, 3 and 1.5 ng) and selected protein spots of A. sylvaticus salivary isolate were excised from 10% gels stained with Coomassie blue G-250. In-gel trypsin digestion and peptide extraction were performed by the modified method described by Shevchenko et al.11 Briefly, bands/spots were manually excised and washed with acetonitrile and AB. The destained gel pieces were dried completely in a vacuum centrifuge (Thermo Scientific, Asheville, North Carolina). Dried gel pieces were rehydrated in a digestion buffer containing 25 mmol L–1 AB and 5 mg L–1 trypsin. The digestion was carried out at 40°C for two hours under 750 revolutions per minute mixing in a Comfort thermomixer (Eppendorf, Hamburg, Germany). The resulting solution surrounding the gel was transferred into a new vial and 10 µL of this free solution were taken for testing by the simplified method (sample A). The tryptic peptides from the remaining gel pieces were extracted with 10 µL of an acetonitrile:water:FA mixture (50 : 45 : 5, v/v) by five minutes sonication. The extract was mixed with the remaining free solution (after removing sample A) for testing by the published method (sample B).
Post-digestion labeling by 18O The peptides were subjected to C-terminal follows:
18
O labeling as
(1) Sample A (simplified method). The sample, in a volume of 5 µL, was mixed with 0.5 µL of 0.5 mol L–1 AB, 1 µL of trypsin (0.1 mg mL–1) and 6.5 µL of H218O. (2) Sample B (published method). The sample, in a volume of 5 µL, was dried in a vacuum centrifuge and then 5 µL of H218O, 3.5 µL of H2O, 0.5 µL of 0.5 M of AB and 1 µL of trypsin (0.1 mg mL–1) were added. In both cases, post-digestion labeling was carried out at 40°C for two hours.
Post-digestion labeling by reductive dimethylation The peptides were subjected to modification of amino groups (N-termini and lysine residues) using a protocol adapted from
A. Voráˇc et al., Eur. J. Mass Spectrom. 20, 255–260 (2014) 257
Hsu et al.17 The digests, in a volume of 5 µL, were mixed with 45 µL of 100 mM acetate buffer (pH = 6), 10 µL of 4% formaldehyde and 10 µL of 1 M sodium cyanoborohydride. After 10 min, the reaction was stopped by adding 10 µL of 4% amonium hydroxide solution, and the mixture was finally acidified by adding 10 µL FA. The peptides were purified and preconcentrated to 10 µL final volume using a ZipTip (U-C18, Millipore) according to the manufacturer’s instructions.
Mass spectrometry Peptide solutions were spotted onto an AnchorChip target (Bruker Daltonics) in a volume of 1 µL and partly dried for approximately 1 minute. Then 0.6 µL of the matrix solution (2 mg mL–1 CHCA in TFA : H2O:ACN mixture 0.8 : 32.5 : 66.7, v/v) was added to the sample and it was allowed to dry. For desalting of the samples, the AnchorChip was washed in 0.5 L of 5% FA for one minute. Mass spectra measurements were performed on an Ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) operating in a reflectron positive mode (accelerating voltage 25 kV) and equipped with a SmartBeam laser. For each analysis, 1000 single laser pulse spectra were accumulated. Seven replicate analyses were performed from each sample. An external calibration procedure was employed, using a mixture of seven peptide standards (Bruker Daltonics) covering the mass range 700–3100 Da. Flex Analysis 3.0 (Bruker Daltonics) software was used for data processing. Protein identification was performed using the Mascot search engine (Matrixscience, London, UK). Data were searched with a mass tolerance of 30 ppm allowed during processing MALDI MS data for PMF (peptide mass fingerprinting) and 0.6 Da during processing LID-LIFT data for MS/MS ion searches. All searches were carried out using the NCBI database without taxonomic restriction. Oxidation of methionine was set as an optional modification and one enzyme miscleavage was set for all searches. The identity of each peptide was verified by an MS/MS ion-search yielding a MASCOT score value >60.
Deconvolution of 16O/18O-containing isotopic spectral patterns To determine the isotopological identity of C-terminal 16O/18Oderived peptides, deconvolution of 16O/18O-containing isotopic spectral patterns was employed, based on an approach described elsewhere.18 As a result, it was possible to give a percentage representation of the isotopologues of C-terminal 18 O-labelled peptides, i.e. the C-terminal carboxylic group containing either 2*16O, 1*16O + 1*18O, or 2*18O.
Results and discussion The difference between this simplified method and the previously published method7 is in eliminating several steps in the workflow, including peptide extraction from a gel, vacuumassisted drying and reconstitution in the labeling buffer. This modification clearly enhanced the entire procedure in terms
Table 1. Sequence coverage (together with confidence limits at a = 0.05) for triplicates (I, II and III) of CKI1 digested with trypsin using the method without (simplified method) and with (published method7) acid extraction of peptides from gels.
Sequence coverage without acid extraction – sample A (%) Sample I, 74 ± 6
Sample II, 78 ± 6
Sample III, 77 ± 6
All samples, 76 ± 3
Sequence coverage with acid extraction – sample B (%) Sample I, 72 ± 7
Sample II, 78 ± 4
Sample III, 74 ± 6
All samples, 75 ± 3
of rapidity and simplicity. Additionally, possible sample losses during vacuum-assisted drying were eliminated. To evaluate the performance of the simplified method, results were primarily compared with those obtained using the published protocol.7 In the first experiment, we tested whether eliminating the extraction step (in the case of in-gel digestion) led to a decrease in protein sequence coverage. In-gel digestion of histidine kinase CKI1 (selected as a model protein yielding a set of tryptic peptides differing in their molecular weight, pI and hydrophobicity) was accomplished in triplicate and the sequence coverages obtained from the free solution surrounding the gel and after peptide extraction from the gel were compared (for details, see Sample treatment). The results are summarized in Table 1. As verified by Student’s t-test (obtained t = 0.286 against P = 2.021 at p = 0.05), sequence coverage was practically the same for both the published and simplified methods. This implies that omitting the acid-extraction step by using the free solution surrounding the gel after digestion did not lead to qualitative loss of particular peptides, as no additional peptides, detectable by MALDI MS, were released from the gel. For the purpose of the estimation of quantitative losses, bands containing an exact amount of BSA were digested. We obtained a detection limit for BSA tryptic peptides equal to 6 ng of BSA loaded onto the gel when the free solution surrounding the gel was applied, and 3 ng after gel extraction was included. The labeling step can thus directly follow proteolytic cleavage without any significant quantitative losses. The extraction step probably cannot be avoided in the case of highly hydrophobic peptides.19,20 In the subsequent experiment, we compared the entire method with the aim of evaluating the efficiency of 18O atom incorporation into the peptide C-terminus. Tables 2(a) and (b) show the most abundant tryptic peptides of CKI1 with representation of particular isotopic forms derived from the mass spectra by deconvolution. Using either the published or the simplified method, two 18O atoms were incorporated by trypsin into the C-terminus of all peptides. As expected, the tryptic peptide located at the protein C-terminus showed no 18O incorporation. The most notable differences between the outputs of the two compared methods were found in cases concerning peptides TPIIAVSGHDPGSEEAR, KPIGNPEDEQETSKPSDDEFLR and ETIQAGMDAFLDKSLNQLANVIR. In these three cases, predominantly one 18O atom was incorporated and only a very low proportion of the peptides were subjected to
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Simplified Method for Peptide de novo Sequencing using 18O Labeling
Table 2a. The list of the most abundant tryptic peptides of CKI1 obtained by the simplified method (sample A, without acid extraction and vacuum-assisted drying) with representation of particular isotopic forms.
Observed
Sequence
2*16O (%)
1*16O, 1*18O (%)
2*18O (%)
24.2 ± 0.8
54 ± 2
22 ± 1
Start
End
101
109
1016.6302
R.LVTEGLTQR.E
182
191
1127.7136
K.SLNQLANVIR.E
34 ± 2
52 ± 1
13.6 ± 0.8
64
74
1276.7529
R.VLVVDDNFISR.K
23.5 ± 0.6
55.7 ± 0.7
20.8 ± 0.9
152
168
1735.9353
R.TPIIAVSGHDPGSEEAR.E
58 ± 1
40 ± 1
2.1 ± 0.7
1
16
1768.9182
-.GSSHHHHHHSSGLVPR.G
29 ± 1
56 ± 2
15 ± 1
84
100
1837.9112
K.MGVSEVEQCDSGKEALR.L
49 ± 11
35 ± 8
16 ± 8
147
168
2298.2388
K.SYGVRTPIIAVSGHDPGSEEAR.E
18 ± 3
44 ± 5
38 ± 5
39
60
2531.3968
R.KPIGNPEDEQETSKPSDDEFLR.G
44.6 ± 0.9
49 ± 1
6.6 ± 0.9
169
191
2547.3200
R.ETIQAGMDAFLDKSLNQLANVIR.E
62 ± 6
29 ± 9
10 ± 7
Table 2b. The list of the most abundant peptides of CKI1 obtained by the published method7 (sample B – with acid extraction and vacuum-assisted drying) with representation of particular isotopic forms.
Observed Start
End
101
109
1016.6302
Sequence R.LVTEGLTQR.E
2*16O (%)
1*16O, 1*18O (%)
2*18O (%)
25 ± 1
55 ± 2
20 ± 2
182
191
1127.7136
K.SLNQLANVIR.E
25.8 ± 0.4
55.2 ± 0.7
19.0 ± 0.5
64
74
1276.7529
R.VLVVDDNFISR.K
24 ± 1
56 ± 1
20 ± 1
152
168
1735.9353
R.TPIIAVSGHDPGSEEAR.E
30 ± 2
56 ± 1
15 ± 2
1
16
1768.9182
-.GSSHHHHHHSSGLVPR.G
25.7 ± 0.8
54 ± 1
20 ± 1
84
100
1837.9112
K.MGVSEVEQCDSGKEALR.L
31 ± 2
52 ± 3
17 ± 3
147
168
2298.2388
K.SYGVRTPIIAVSGHDPGSEEAR.E
51 ± 6
35 ± 8
14 ± 9
39
60
2531.3968
R.KPIGNPEDEQETSKPSDDEFLR.G
21.2 ± 0.7
53 ± 1
26 ± 1
169
191
2547.3200
R.ETIQAGMDAFLDKSLNQLANVIR.E
29 ± 5
48 ± 6
23 ± 3
double labeling by the simplified method. We also observed that peptides differed in their degree of 18O incorporation, which is a phenomenon already described in the literature.18 Nevertheless, for all peptides (except the C-terminal one) and both methods, a two-hour labeling resulted in a notable change in the isotopic pattern of the peptide [M+H]+ signal in the MS mode, and for all y-series signals in the MS/MS mode. The results thus demonstrate that there was no significant difference in peptide-sequence information obtained by either method. The simplified 18O-labeling was also compared to reductive dimethylation to assess the method performance in peptide de novo sequencing. The parameter for the assessment of these two approaches was the number of labeled MS/MS fragments (y-series for 18O-labeling and b-series for dimethylation) seen from the spectra of ten dominant peptides from three BSA tryptic digests. The MS/MS sequence coverage for unlabeled peptides was 85 ± 2% for the y-series and 76 ± 5% for the b-series. After 18O-labeling, 71 ± 1% of y-fragments were observed, while after reductive dimethylation we found
68 ± 3% of all theoretical b-fragments. The relatively greater decrease in sequence coverage after 18O-labeling in comparison to unlabeled peptide analysis probably stems from a decrease in the signal intensity, caused by distribution of the signals into more peaks. On the other hand, the specific isotopic pattern of the labeled fragments greatly facilitated their assignment. The most prominent advantage of dimethylation is in distinguishing between lysine and glutamine residues. Unfortunately, apart from the peptide N-terminus, dimethylation also modifies lysine residues frequently located at the C-terminus of tryptic peptides, which complicates the interpretation of their MS/MS data. A combination of data from both labeling approaches would, certainly, enable de novo data interpretation with the highest confidence. The suitability of the simplified method for the determination of peptide sequence de novo was tested on salivary protein isolates from A. sylvaticus, representing an organism with an unsequenced genome and a limited number of protein sequence entries in the databases. After in-gel digestion and MALDI-MS/MS analysis, the samples that yielded
A. Voráˇc et al., Eur. J. Mass Spectrom. 20, 255–260 (2014) 259
Figure 1. MALDI-MS/MS spectra of a 1793.8 Da tryptic peptide obtained after digestion of the A. sylvaticus salivary protein. The MS/MS spectra of 18O-labeled peptide (a) show that all detected y-ion fragments ions possess a characteristic peak shifted by +2 Da corresponding to the 18O tag in the peptide C-terminus (marked with an asterisk) in contrast to the unlabeled peptides (b).
no significantMASCOT scores against the NCBI database were treated according to the simplified procedure. Almost all peptides in the digests were modified by one or two 18O atoms, as was confirmed from the characteristic change in their isotopic patterns. The exceptions usually corresponded to one unlabeled peptide per digest, probably due to its localization at the protein C-terminus. The labeled peptides were then subjected to repeated MALDI-MS/MS analysis. Similarly to the example shown in Figure 1, the y-ions in all examined peptides were highlighted by a clearly visible change in the isotopic pattern, while other MS/MS fragment ions remained unlabeled. The observation of a characteristic isotopic pattern then notably facilitated the MS/MS data interpretation, resulting in successful peptide de novo sequencing. In the case of the peptide selected for Figure 1, the BLAST search clearly assigned its de novo determined sequence (pETPENLVFYSENVDR, where pE stands for pyroglutamic acid, and L cannot be distinguished from I) to a homologous segment (ATSENLVFYDENVDR) present in Mus musculus odorant binding protein Ib (NCBI entry gi|123121834).
Conclusions We have described a method for peptide de novo sequencing based on direct labeling of tryptic peptides by the addition of H218O and trypsin. Elimination of peptide extraction from gels, vacuum-assisted drying, and redissolution of the digests were found not to decrease sequence coverage or the possibility of distinguishing C-terminal fragment ions as compared to the published method.7 The time required for the new protocol was notably reduced and the analysis workflow was significantly simplified compared to the published method.
Acknowledgments This work was supported by the project “CEITEC – Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund and by project No. P206/12/G151 of the Czech Science Foundation. P. Stopka, from Department of Zoology, Faculty of Science, Charles University, is gratefully acknowledged for kindly providing A. sylvaticus salivary protein samples. Colleagues from the research group Functional Genomics and Proteomics of Plants, of our institute, are gratefully acknowledged for kindly providing the CKI1 protein sample. English language was kindly revised by Prof. J. D. Brooker.
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