Reagents for Isobaric Labeling Peptides in Quantitative Proteomics

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Technical Note

A Novel Reagent for Isobaric Labeling Peptides in Quantitative Proteomics Yan Ren, Yanbin He, Zhilong Lin, Jin Zi, Huanming Yang, Shenyan Zhang, Xiaomin Lou, Quanhui Wang, Shuwei Li, and Siqi Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00321 • Publication Date (Web): 27 Sep 2018 Downloaded from http://pubs.acs.org on September 28, 2018

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Analytical Chemistry

A Novel Reagent for Isobaric Labeling Peptides in Quantitative Proteomics Yan Ren1, Yanbin He1, Zhilong Lin1, Jin Zi1, Huanming Yang1,2, Shenyan Zhang1, Xiaomin Lou3; Quanhui Wang4, Shuwei Li5*, Siqi Liu1* 1

BGI-Shenzhen, Beishan Industrial Zone 11th building, Yantian District, Shenzhen,

Guangdong, 518083, China, 2

James D. Watson Institute of Genome Sciences, Hangzhou 310008, China

3

Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101,

China; 4

Beijing Protein Innovation, B-8, Beijing Airport Industrial Zone, Beijing, 101318,

China 5

Institute for Bioscience and Biotechnology Research, University of Maryland

College Park, Rockville, MD 20850;

*To whom correspondence should be addressed: Siqi Liu, BGI-Shenzhen, Beishan Industrial Zone 11th building, Yantian District, Shenzhen,

Guangdong,

518083,

China.

Tel:

86-755-36307403;

E-mail:

[email protected]

Shuwei Li, Institute for Bioscience and Biotechnology Research, University of Maryland College Park, Rockville, MD 20850; E-mail: [email protected] 1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Currently, the commercial reagents for isobaric peptides labeling (TMT and iTRAQ) have some drawbacks, such as high cost in experiments especially in quantitation for the modified peptides and inconvenient handling for variable sizes of samples. Herein, we developed a set of 10-plex isobaric tags (IBT) with high stability and low cost. The labeled peptides were sensitively detected on Orbitrap Q Exactive MS with MS2 resolution of 35,000 at 30% NCE, while the peptides were efficiently labelled over 97% by IBT at ratio of 10:1 of reagent/peptide (w/w) in 200 mM TEAB buffer for 2h. The IBT labeling was demonstrated with a wide dynamic range of 50 folds without obvious matrix effect on quantification. Importantly, there was little quantification bias found among the individual IBT tags, indicating that the peptides labeled by different tags were quantitatively comparable. The IBT 10-plex reagents were applied for dynamically monitoring the quantitative responses of phosphoproteome stimulated by EGF treatment in Hela cells. In total, 5,361 unique phosphopeptides were identified, which reached a similar conclusion as other reported. The IBT reagents were therefore experimentally proven as a new type of reagents for isobaric peptides labeling and useful in a large quantity peptides of quantitative proteomics.

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Introduction There are several approaches based upon mass spectrometry for quantitative proteomics, including label-free, MS1- and MS2-based1. Isobaric tag labeling methods that belong to MS2-based approaches are widely used2-6. Compared with label-free data dependent analysis (DDA) methods, the analysis time and run-to-run variations in the isobaric labeling are greatly reduced because the labeled peptides from multiple samples are mixed together before LC-MS/MS assay. The sensitivity of peptide detection is greatly enhanced because the MS/MS signal(s) for a peptide is contributed by multiple samples so that its correspondent signal intensity is strengthened. More importantly, isobaric labeling methods minimize inconsistent peptides identified among different samples, which remains a common problem in label-free workflow. Compared with MS1-based methods such as stable isotope labeling using amino acids in cell culture (SILAC), isobaric peptide labeling approaches reduce the complexity of chromatographic separation for peptides, especially for multiple samples. In addition, SILAC-based quantitation relies on the incorporation of isotopic arginine or lysine into newly synthesized proteins, which is easily achievable in cultured cells but more difficult with tissue samples and bacteria7, whereas isobaric labeling would not be limited by sample sources. Apparently, isobaric approach is one of the most popular methods in proteomic quantification at large scale.

Similar to other approaches, isobaric labeling quantification has some inherent drawbacks. Pichler et al systematically compared of the quantitative results in the different isobaric labeling approaches8. In general, these commercial reagents are prohibitively expensive, especially for applications that require labelling large quantity of peptides9,10. For instance, quantification of PTM often requires specific enrichment of modified peptides after initial peptide labeling with isobaric reagent to eliminate variations introduced by sample handling. It is well known that tyrosine 3



phosphorylation (pTyr) is a very important PTM that plays critical roles in cell growth and differentiation11,12, whereas pTyr modification is very scarce (0.5-4% of all phosphorylation events). Thus a large amount of starting material (about 10 mg of proteins for mammalian cells) is often required in order to enrich enough pTyr peptides for MS detection. Labeling such high quantity of peptides with isobaric reagents could become an economic burden in many laboratories. Besides, most commercially available isobaric labeling reagents is based on amine-reactive NHS ester that is prone to hydrolysis. This means a vial with isobaric labeling reagent after dissolving must be used completely as soon as possible. Such restriction often causes inconvenient operation during experiment.

To circumvent these problems, we have developed 10-plex isobaric tags (IBT) with high stability and low cost, which are the improved version of 6-plex DiART reagents previously reported by our laboratories13,14. By utilizing the minor mass difference (6.3mDa) between the isotopic pairs of 13C/12C and 15N/14N atom (s), we were able to expand the labeling groups from 6 to 10. To evaluate the IBT reagent, the parameters of mass spectrometer and the labeling efficiency were systematically optimized. Furthermore, we quantitatively mapped dynamic phosphoproteome responses in Hela cells to EGF stimulation, and firmly concluded IBT as a new isobaric labeling tag for quantitative proteomics at large scale.

Materials and Methods Synthesis of the IBT reagents The synthesis of IBT 10-plex reagents was slightly modified from our previous reports13-15. Most isotope starting materials were purchased from Cambridge Isotope Laboratories (Andover, MA), including 1-13C Leucine, 2-13C Leucine, 1-13C, 15N Leucine, 2-13C, 15N Leucine, 15N Leucine, 1,2,3-13C3, 15N Alanine, 1,2,3-13C3 Alanine, 2,3-13C2 Alanine, 1-13C, 15N Alanine, 15N Alanine, and 13C Formaldehyde (20% in water). The 5,5-13C2 Leucine used in the synthesis of T-118C 4


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and 5,5-13C2, and 15N Leucine used in the synthesis of T-119 were prepared based on a previously reported method16. IBT 10-plex is currently commercialized by Nanjing Apollomics Biotech Co. (www.apollomics.com).

IBT labeling peptides Right before labeling peptides, IBT precursors were treated with equal mole ratio of TSTU (1,1,3,3-tetramethyl-O-(N-succinimidyl) uronium tetrafluoroborate) purchased from TCI (Shanghai, China) in isopropanol to final concentration at 25µg/µL and were incubated at room temperature for 10 min. The activated IBT was mixed with a certain amount of peptides that was dissolved in 0.2M triethylammonium bicarbonate (TEAB). In the labeling reaction, isopropanol concentration was remained at >75%, and the labeling process was stopped by adding trifluoroacetic acid (TFA) after incubation at ambient temperature for 2h.

Detection of the peptides labeled with IBT IBT labeled peptides were detected by an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA) coupled with a nano reverse phase (RP) column (5-µm Hypersil C18, 75 µm × 150 mm, Thermo Fisher Scientific, Waltham, MA) as described before with setting in positive ion mode and data-dependent manner with full MS scan from 350-2,000 m/z, resolution at 70,000, MS/MS scan with minimum signal threshold 20,000, and isolation width at 2 Da except with a MS/MS resolution of 35,000. The MS2 noise cutoff was applied at S/N of 1.5 and the signals of reporter ions at least 5% intensity of the maximum peak were accepted for quantification.

EGF stimulation to the cultured Hela cells Hela cells purchased from ATCC were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. A concentration of 150 ng/mL EGF was used to treat Hela cells for 1, 5, 10, 15, 20, 30, 40, 50 and 60min in a serum free 5



medium, respectively. The cells with different EGF stimulation periods were harvested by centrifugation and were washed twice with PBS to remove the EGF remaining.

Hela cell protein extraction, digestion and peptide labeling The Hela cell protein extraction, digestion and peptide desalting were performed as described before. The peptides (300µg) from the treated Hela with different EGF incubation periods were labeled with IBT in a w/w ratio of 1:10 as mentioned above and mixed for the following phosphopeptide enrichment.

Enrichment and fractionation of phosphopeptides The IBT-labeled phosphopeptides were enriched by TiO2 beads following the previous published protocol17 and fractionated by a high pH Reversed-Phase Peptide Fractionation Kit (20mg, Thermo Scientific, Rockford, lL) using a stepwise elution with different ACN concentrations (5, 7, 8, 9, 10, 11, 13, 15, 17, 20, 25, and 50% ACN in trimethylamine). The 12 fractions were combined into 6 fractions for peptide separation and identification using LC-MS/MS.

Database searching Protein identification and quantification were performed using iQuant software with iPeak searches integrated by three search engines (MyriMatch v2.2.10165, X!Tandem v2017.2.1.2 and MS-GF+ v2017.01.13)18,19. The peptide identification was conducted against the SwissProt human database (released on 2017_04) combined with decoy sequences. The false discovery rate (FDR) was set to less than 1% for both peptide and protein identification. Trypsin was selected as the specific enzyme with a maximum of one missed cleavages per peptide. Fixed modifications included IBT 10-plex (N-term), IBT 10-plex (K), carbamidomethylation (C) and variable modifications included oxidation (M), deamidatioin (N, Q), IBT 10-plex (Y), and phosphorylation (S, T, Y). Data were searched with peptide mass tolerance at 20 ppm 6


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and fragment mass tolerance at 0.1 Da. Only were those phosphosites with more than 0.75 phsophoRS score considered as genuine phosphosites20. The pval of phosphopeptide quantification was calculated following the Cauchy distribution then was transferred into qval by Benjamin-Hochberg algorithm21,22. For the labeling efficiency evaluation, the setting of database searching was as same as above except that the modification of IBT 10-plex (N-term and K residue) was set as variable.

Results and Discussion Design of IBT We previously reported DiART 6-plex tags (nominal mass 114-119) for concurrent quantification of up to six samples. In this study, we expanded 6-plex DiART to 10-plex IBT by carefully placing 13C and 15N into the slightly modified DiART structure (Fig. 1) 12. This design relies on high-resolution MS instruments that can distinguish neighboring reporter ions with small mass difference. We also keep the IBT molecules as free acid precursors, which are quite stable and easy for handling. These precursors can be treated with TSTU for one-step conversion to NHS esters, then the activated IBT tags are ready for peptide labeling. The 12-plex DiLeu was reported as a new labeling reagent23. The chemical structures of DiART and 12-plex DiLeu are similar, whereas the two reagents contain different isotope types. The design of IBT follows the same DiART structure, yet only uses 13C and 15N isotopes, which could prevent the unpredictable deuterium-associated chromatographic shift to guarantee the quantitation accuracy\.

Optimization of MS parameters for detection of the peptides IBT-labeled In labeling-based proteomic quantification, it is necessary to balance the better separation of neighboring reporter ions (e.g. 115C vs. 115N) and the more detection of peptides24. We thus tested the effect of MS2 resolution on the identification of labeled peptides using the tryptic digests of α-casein (Supplementary Fig. S1A and 1B) and reached the same conclusion with other reports that MS2 resolution at 35,000 was 7



suitable for balancing reporter peak separation and peptide identification10.

The normalized collision energy (NCE) for 6-plex DiART labeled peptide fragmentation was only optimized on an LTQ-Obitrap Velos. Here we optimized the optimal NCE setting between 24% and 36% to balance the identification of peptides by enough fragments and the quantification of IBT labeled peptides from the intensity of reporter ions on an Orbitrap Q-Exactive. As shown in Supplementary Figure S1C and 1D, NCE at 30% therefore was proved to achieve optimum identification and quantification of IBT-labeled peptides.

Optimization of reaction conditions for IBT labeling peptides Efficient labeling of peptides is a prerequisite for accurate quantification of proteins, which is generally presented as the percentage of identified labeling-peptides to total identified peptides. To systematically optimize the IBT labeling conditions, the TEAB concentrations used for peptide suspension and labeling, the ratio between labeling reagents and peptides, and the labeling time were valuated. As shown in Supplementary Figure S2A-C, the condition that peptides were suspended and labeled in 200mM TEAB solution with a ratio of 10 for IBT reagents to peptides for 2h was found optimal for IBT labeling.

Commercially available labeling reagents such as iTRAQ and TMT are activated NHS esters that are easily hydrolyzed once dissolved in solution, and are completely lost labeling activity within a short period3-5. Once vials of these reagents are opened, all of them have to be used in one experiment. Thus, the hydrolysis properties would restrict an experimental design and cause the increased cost in experiments. We designed an in-situ activation of IBT precursors right before peptide labeling and found that 95% labeling efficiency maintained even after six months storage in non-activated status (Supplementary Fig. S2D). Even though the activated IBT is kept at -80°C freezer, the labeling efficiency still remained at 90% after 1 day. This 8


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suggests that the free acid IBT precursors provide more flexible use for peptide labeling.

Evaluation of relative quantification based on the peptides IBT-labeled For the sake of appraising the quantification bias, the tryptic peptides with equal amount from HeLa cells were labeled with each tag respectively, then the labeled peptides were pooled and delivered to mass spectrometer for peptide identification and quantification. As demonstrated in Fig. 2A, the ratios of all the other 9 channels over 114 are close to 1.0 with acceptable CV values, suggesting there is little in quantitation bias among different tags.

The dynamic range of IBT quantification was evaluated from two aspects based on the same sample to overcome the ratio compression due to co-elution from different samples. In the first experiment, the tryptic peptides of BSA were separately labeled with

individual

IBT

tags

and

were

mixed

with

certain

ratios

at

114:115N:115C:116N:116C:117N:117C:118N:118C:119 = 1:3:9:25:50:1:3:9:25:50. The relative abundance was estimated with IBT 116C as a reference. As illustrated in Fig. 2B, the intensities of reporter ions appear a linear correlation to the theoretical values within the range up to 50 folds (slope = 1.10 and R2 = 0.97). Closely checking the abundance ratios at relatively low range from 1, 3 and 9 (114, 115N, 115C, 117N, 117C and 118N/116C), the data on Fig 2B inset indicates that the values of slope and R2 are acceptable for protein quantification (slope = 0.87 and R2 = 0.91). In the second experiment, the tryptic peptides of HeLa cell lysates were separately labeled by

each

IBT

and

were

mixed

at

different

ratios

at

114:115N:115C:116N:116C:117N:117C:118N:118C:119 = 3:5:10:1.5:1:1:1.5:10:5:3. The relative abundance was estimated with IBT 117N as a reference. As shown in Fig. 2C, most of the median ratios are close to their expected values. These evidences endorse that IBT is appropriate in relative quantification proteomics in either simple or complex system. 9



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It is well known that quantification of complex samples with isobaric tags can undergo a level-off effect, in which potentially large intensity differences are suppressed by matrix background25-27. To check whether the IBT signals are influenced by matrix, we compared the relative quantification of BSA peptides in the matrix with/without E.Coli peptides. The tryptic peptides of BSA were individually labeled

by

IBT

reagents

and

mixed

with

a

ratio

of

114:115N:115C:116N:116C:117N:117C:118N:118C:119 = 1:2:4:8:10:1:2:4:8:10 or labeled together with the same amount of E. Coli peptides in an incorporation ratio of 1:2:4:8:10:1:2:4:8:10 for BSA, which were then mixed in equal amount of total peptides for LC-MS/MS analysis. In the group without E.Coli peptides, the quantification displays a linear correlation with slope at 0.83 and R2 of 0.97, whereas in the group with E.Coli peptides, the linear fitting exhibits slope at 0.45 and R2 at 0.96 (Fig. 2D). Obviously, the complex background exerts a serious matrix effect to the tag MS signals. However, the relative quantification remains a linear correlation even though the tag MS signals are globally attenuated. IBT thus is expected to be feasible in relative quantification in a system with complex peptides.

Quantitative phosphoproteome responses to EGF treatment in HeLa cells monitored by IBT. Since IBT enables the simultaneous quantification of up to 10 different samples, we adopted the new reagents to record the time-dependent (1, 5, 10, 15, 20, 30, 40, 50, 60 min) phosphoproteome changes in an EGF-stimulation system. Altogether, a total of 5361 phosphopeptides with 4882 confidently localized phosphosites in 1971 proteins were identified (Supplementary Table S1).

Mann’s group implemented quantified phosphoproteomes of Hela cells treated with EGF within 20 min using SILAC quantitation28, in which the dynamic phosphopeptides derived from SILAC were clustered into 6 patterns. For a period of 10


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20 min after EGF treatment, 133 phosphoproteins with significant abundance changes were identified by both approaches, SILAC and IBT, in which 42 proteins (Supplementary Table S2) shared the same patterns of abundance change. As presented in Fig. 3A, the IBT labeled dynamic phosphoproteomics within 20 min is clustered to 9 patterns using the fuzzy c-means algorithm embedded in the Genesis software29, 8 of them being in agreement with the SILAC patterns. It has been suggested that EGF binds with dimerized epidermal growth factor receptor (EGFR), which leads to the autophosphorylation of Y1172 on EGFR and rapidly initiates the signaling cascade to its protein substrates such as SHIP2, PLCG1, SHC1 and MAPKs. We also observed that the abundance of the phosphopeptides from these proteins rapidly increased within a short time and remained at the same level during the rest time of the experiment (Fig. 3B). Besides, we extended the EGF treatment in Hela cells to 60 min and observed a new pattern consisting of 118 phosphopeptides from 102 proteins (Supplementary Table S3). Obviously, the dynamic curves based upon these quantified phosphoproteins with IBT provide a strong evidence to support the hypothetic model of EGF activation. Moreover, the quantitative measurements of phosphopeptide conducted by the two approaches reach the same conclusion to elucidate the mechanisms of EGF stimulation.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31500670) and ShenZhen Engineering Laboratory for Proteomics (DRC-SZ [2016]749).

Figure Legends Figure 1. The scheme of IBT chemical structure. Structure and application of IBT-10plex reagents. (Top) The IBT-10plex reagents were activated by TSTU, then reacted with peptides. Labeled peptides were analyzed by LC-MS/MS to generate reporter ions for peptide quantification. (Bottom) Red and 11



green asteroids represent the positions of 15N, 13C, respectively. The monoisotopic mass of each reporter ion is shown in parenthesis.

Figure 2. Evaluation of the precision and accuracy of IBT labeling. A) Analysis of quantification bias using individual IBT reagents to label the same peptides B) Quantification of BSA peptides that were pre-mixed at different ratios (Y-axis) verus their therotical ratios (X-axis). The inset is the expanded area with low ratios. 11(X) stands for the other nine reporter ions except 116C as indicated. C) Standard deviation of BSA peptides quantification that were pre-mixed at different ratios (1:1, 2:1, 5:1, 10:1). D) Matrix effects on IBT quantification. W/O spike-in means BSA peptides ratios were meansured in a clean background. Spike-in means BSA peptides ratios were measured by mixing BSA peptides with E.Coli digests.

Figure 3. Dynamic changes of phosphoproteomics in response to EGF in Hela cells measured by IBT. A) Nine different dynamic patterns of phosphopeptide in responses to EGF treatment of HeLa cells within 20 min. This clustering analysis was based on the fuzzy c-means algorithm. B) Dynamic changes of representative proteins involved in EGFR-triggered pathways upon EGF treatment of HeLa cells within 60 min.

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2.5

SHC1

2

EGFR SHIP2

1.5 1 0.5 0

1

5

10 15 20 30 40 50 60

2 MAPK1 MAPK7 1.5

MAPK3

Cluster9

Cluster8

1

0.5 0

1

5

10 15 20 30 40 50 60

Time(min) Time(min)

Figure 3



338x190mm (96 x 96 DPI)


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338x190mm (96 x 96 DPI)


Reagents for Isobaric Labeling Peptides in Quantitative Proteomics

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