Improved 6-Plex Tandem Mass Tags Quantification Throughput Using

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Improved 6-plex TMT quantification throughput using a linear ion trap – HCD MS scan 3

Jane May Liu, Michael J. Sweredoski, and Sonja Hess Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01067 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 6, 2016

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Analytical Chemistry

Improved 6-Plex TMT Quantification Throughput Using a Linear Ion Trap – HCD MS3 Scan Jane M. Liu,1,2* Michael J. Sweredoski,2 Sonja Hess2* 1

Department of Chemistry, Pomona College, Claremont, California 91711, United States Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, California Institute of Technology, Pasadena, California 91125, United States

2

KEYWORDS: mass spectrometry, protein quantitation, isobaric tags, TMT, quadrupole ion trap, Orbitrap

ABSTRACT: The use of tandem mass tags (TMT) as an isobaric labeling strategy is a powerful method for quantitative proteomics, yet its accuracy has traditionally suffered from interference. This interference can be largely overcome by selecting MS2 fragment precursor ions for highenergy collision induced dissociation (HCD)-MS3 analysis in an Orbitrap scan. While this approach minimizes the interference effect, sensitivity suffers due to the high AGC targets and long acquisition times associated with MS3 Orbitrap detection. We investigated whether acquiring the MS3 scan in a linear ion trap with its lower AGC target would increase overall quantification levels with a minimal effect on precision and accuracy. Trypsin-digested proteins from Saccharomyces cerevisiae were tagged with 6-plex TMT reagents. The sample was subjected to replicate analyses using either the Orbitrap or the linear ion trap for the HCD-MS3 scan. HCD-MS3 detection in the linear ion trap vs. Orbitrap increased protein identification by 66% with minor loss in precision and accuracy. Thus, the use of a linear ion trap – HCD MS3 scan during a 6-plex TMT experiment can improve overall identification levels while maintaining the power of multiplexed quantitative analysis.

In recent years, the use of isobaric chemical tags (e.g. tandem mass tags (TMT)) in quantitative proteomics has allowed for increased multiplexing in a wide-variety of experiments.1-6 This approach uses NHS chemistry to label primary amines.6 As each and every peptide has a primary amine at its Nterminus, theoretically all peptides in a sample should be labeled at least once by a chemical tag. The labels have the same mass such that identical peptides from different samples each labeled with a unique tag will produce identical MS1 precursor ions. After fragmentation, the labels release unique reporter ions that are used for quantitation. Duplex, 6-plex and 10-plex TMT-labeling reagents are commercially available and in a recent side-by-side comparison, it was observed that TMTlabeling can afford results that are more precise and accurate than other stable isotope labeling-based proteomics methods such as “stable isotope labeling with amino acids in cell culture” (SILAC) or dimethyl labeling.7 The use of TMTlabeling, however, generally suffers from interference from co-isolated precursor peptides that limit the quantification capabilities of the approach.7,8 An elegant way to overcome the co-isolation induced interference was recently proposed, in which MS2 fragment ions are selected for high-energy collision induced dissociation (HCD)-MS3 analysis, using the Orbitrap for quantification of the TMT reporter ions.8 This secondary HCD-based fragmentation significantly reduces any interference.8 Sensitivity suffers, however, and extensive fractionation (e.g. 20 fractions)

was used to obtain reasonable protein identification and quantification numbers.8 A further improvement of this method was the introduction of synchronous precursor selection (SPS), where isolation waveforms with multiple frequency notches were used to fragment multiple MS2 precursors at once, thereby improving sensitivity.9 However, overall sensitivity remained relatively low. In our preliminary analysis of the samples acquired using the MS3 Orbitrap quantification method with SPS9 we found a significant portion of the acquisition time was devoted to MS3 ion injection. We reasoned that the sensitivity of these analyses suffers due to the high AGC targets and long acquisition times associated with MS3 Orbitrap quantification. We sought to lower the AGC target (and thus injection time) by taking advantage of one feature of the new tribrid instruments such as Orbitrap Fusion/Lumos that allows for quantification of TMT reporter ions generated through HCD-fragmentation in a linear ion trap. We hypothesized that the decreased acquisition time associated with detection of the MS3 scan in the linear ion trap would increase quantification throughput with a minimal effect on precision and accuracy. In this report, we compared the identification numbers, accuracy and precision of linear ion trap-based quantification vs. Orbitrap-based analyses on samples labeled with the six different channels of the TMT 6-plex system. We improved the previously optimized TMT method that makes use of SPS and an MS3 scan to minimize interference; the only change we

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implemented was performing the detection of the MS3-HCD generated fragments in the linear ion trap, rather than the Orbitrap. In this manner, we specifically explored the effect that site of MS3 ion detection has on the sensitivity of a quantitative multiplexed experiment. Overall, we observed that HCDMS3 detection in the linear ion trap increases protein identification in 6-plex experiments by 66% with minor loss in precision and accuracy as compared to using the Orbitrap for the MS3 scan. Thus, we are introducing a simple alteration to the common method for acquiring 6-plex TMT-labeled proteomics data that will increase protein identification rates without compromising quantification accuracy. EXPERIMENTAL SECTION Sample Preparation. Protein extract (200 µg) from Saccharomyces cerevisiae was digested with Lys-C and trypsin on Amicon Ultra-centrifugal filter units (10 K molecular weight cutoff) as previously reported.10-13 Digested peptides were desalted by HPLC using a C18 Macrotrap (Michrom Bioresources) (buffer A: 0.2% formic acid in H2O; buffer B: 0.2% formic acid in acetonitrile), followed by concentration in vacuo. Aliquots of tryptic digest (4 µg, each) were used for peptide labeling with TMT 6-plex reagents (Thermo Fisher Scientific) according to the manufacturer’s protocol. The crude reactions of the labeled peptides were mixed 10:2:1:1:2:10, with respect to the reporter ions used (126, 127, 128, 129, 130, 131 m/z, respectively) and the combined mixture was desalted as above. Additional details can be found in the Supporting Information. Nanoflow Liquid Chromatography Tandem Mass Spectrometry. To avoid variations that could arise from alterations in instrument performance, all measurements that were directly compared to each other were carried out on the same LTQ Orbitrap Fusion instrument using the same processed samples, the same nano-HPLC and an identical LC gradient. All experiments were performed on an Orbitrap Fusion with an ancillary nanoLC (EASY-nLC II) (Thermo Fisher Scientific). All columns (20 cm x 50 µm) were packed in-house

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with ReproSil-Pur C18AQ 1.9 µm resin (120 Å pore size, Dr. Maisch GmbH, Ammerbuch, Germany). Solvent A was 97.8% H2O, 2% acetonitrile, 0.2% formic acid; solvent B was 19.8% H2O, 80% acetonitrile, and 0.2% formic acid. The column was enclosed in a column heater operating at 75 °C. After loading, the peptides were separated with a 120-min gradient at a flow rate of 300 nL/min. The gradient was as follows: 2-6% solvent B (7.5 min), 6-25% solvent B (82.5 min), 25-40% solvent B (30 min), 40-100% solvent B (1 min) and 100% B (9 min). The Orbitrap Fusion was programmed in the data dependent acquisition mode for both the MS2 and MS3 scans. An MS1 survey scan of 400-1500 m/z in the Orbitrap at a resolution of 120,000 was collected with an AGC target of 2.0e5 and maximum injection time of 50 ms. Precursor ions were filtered according to monoisotopic precursor selection, charge state (+2 - +7), and dynamic exclusion (70 s with a ± 10 ppm window). The ten most intense MS1 precursor ions were subjected to CID fragmentation (0.5 m/z window, AGC target of 4.0e3, maximum injection time of 150 ms, CE 35%). Following fragmentation, the MS2 precursor population was selected using the SPS waveform (10 notches) and then fragmented by HCD (CE 55%). When the Orbitrap was used for the MS3 scan, the AGC target was 5.0e4 with a maximum injection time of 250 ms (profile mode). When the linear ion trap was used for the MS3 scan, the AGC target was ranged from 1.0e3 to 1.0e4, with a maximum injection time of 250 ms (centroid mode). Data Analysis. MSGF+ (v. 20140630)14 was used to search the raw data and reporter ion intensities were corrected for isotope impurities. Raw data and search results were uploaded to ProteomeXchange with accession: PXD004093. Additional details can be found in the Supporting Information. To assess the accuracy and precision of the data, for each spectrum the fractional intensity of each reporter ion was determined and then compared to the idealized values (e.g. based on the mixing of labeled peptides in a ratio of 10:2:1:1:2:10). The difference between the experimental and the ideal was calculated as a percent error. For quantitative data, graphs were prepared and analyses were performed using GraphPad Prism (v. 6).

Figure 1. Example spectra for the same peptide analyzed using the linear ion trap (left) and the Orbitrap (right) for the MS3 scan


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RESULTS AND DISCUSSION We set out to evaluate whether using a linear ion trap for the MS3 scan rather than an Orbitrap could increase overall performance in a TMT experiment. To begin, we determined an appropriate AGC target value for the MS3 scan in the linear ion trap. While keeping all other scan parameters the same, we tried three different AGC targets for the MS3 scan in the linear ion trap: 1.0e3, 5.0e3 and 1.0e4. Overall, the number of peptide-spectrum matches (PSM), peptides and proteins identified and quantified using an AGC target of 1.0e3 was higher than those observed with either of the other two AGC target values (Figure S1 and Table S1). However, using an AGC target of 5.0e3 or 1.0e4 provided quantification values that were more accurate and precise, when compared to idealized values (Table S1 and Figure S2). To balance overall identification with accuracy and precision, an AGC target of 5.0e3 was applied in all subsequent analyses when the MS3 was performed in the linear ion trap. To compare the results of a TMT 6-plex experiment in which the MS3 scan was acquired in the linear ion trap vs. the Orbitrap, one TMT-labeled sample was used for all analyses. A tryptic digest of yeast whole cell lysate was aliquoted into equal amounts and each aliquot was labeled with one of the six channels of the TMT 6-plex reagent (which generate either 126, 127, 128, 129, 130, or 131 m/z reporter ions) followed by mixing of all six channels in a ratio of 10:2:1:1:2:10, respectively. The Orbitrap- and linear ion trap-MS3 analyses were interleaved, back-to-back with 250 ng of total labeled peptide injected for each analysis. The instrument methods used were identical to the SPS MS3 method previously reported that successfully minimizes interference.9 To make a comparison between the Orbitrap and linear ion trap quantification of the MS3-HCD fragments, half of the samples were quantified in the linear ion trap, the other half was quantified in the Orbitrap. The MS3 spectra of the same peptide, obtained from an ion trap- and an Orbitrap-MS3 scan are shown in Figure 1.

A

B

Figure 2. (A) Number of peptide-spectrum matches (PSM) and unique peptides that were quantified in all reporter channels. (B) Number of proteins that were quantified in all reporter channels. Error bars represent the standard deviation of five technical replicates.

A

B

Figure 3. Accuracy and precision of (A) peptide and (B) protein quantification when collecting the MS3-HCD scan using the linear ion trap (dark blue, left) or the Orbitrap (light blue, right). The proteins analyzed were all identified by >1 peptide. Boxplots indicate the median (middle line), the interquartile range (box) and the 5th and 95th percentile for each experiment (which includes 5 technical replicates). The dashed lines indicate the values for the idealized fractional intensities.

Comparing the intensities of the six reporter channels in each scan suggests that the both methods produce the expected reporter ion profiles. The use of five technical replicates allowed for determination of the reproducibility of the two methods used. The data described below represent the averages and standard deviations observed across five technical replicates. In terms of total PSM and protein identification, the use of the ion trap for the MS3 scan lead to higher numbers than use of the Orbitrap (Tables S2 and S3). In a multiplexed experiment, data analysis, including normalization and quantitative comparisons across all samples, typically requires quantified intensities in all reporter channels. On average, 95% of all quantified spectra had reporter ions in all six channels when the MS3-HCD scan was detected in the ion trap; 70% of spectra were quantified in all six channels when the MS3-HCD scan took place in the Orbitrap (Figure S3 and Table S2). Thus, for all subsequent analyses, we only considered those spectra that corresponded to peptides observed in all six reporter channels. The use of the linear ion trap for the MS3 scan led to 91% more PSM and 84% more unique peptides quantified compared to the Orbitrap-MS3 scans (Figure 2A). This increase in peptide identification, furthermore, corresponds to a 66% increase in protein quantification (Figure 2B). These results support our hypothesis that the use of the linear ion trap for the MS3 scan would allow for increased sensitivity in a TMT-experiment. We then turned our attention to the accuracy and precision of measurements obtained using the linear ion trap for HCD-



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MS3 scans. In comparing the fractional intensities of the reporter ions, we observed that while the averages were roughly the same in both datasets, the range of errors was slightly larger when the MS3 scan was collected in the linear ion trap (Figure 3 and Figure S4). Both linear ion trap and Orbitrap scans led to minor deviations from idealized values for the reporter ion ratios. Although we applied the SPS waveform in selecting the MS2 precursor population for the MS3 scan,9 our results suggest that we may still be observing a minor compression effect in which the fractional intensities of the reporters shift toward unity. Alternatively, these results could be a factor of a negligible mixing error that occurred when the different TMTlabeled peptides were combined. The increase in error associated with data collected with the linear ion trap-HCD MS3 scans, compared to the Orbitrap scans, is small, but consistent (Figures 3 and 4). In looking at quantification of proteins identified by >1 peptide across the six reporter channels, the median percent error (averaged across the five replicates) for the ion trap scans was 5.5, compared to 3.8 for the Orbitrap scans (p < 0.0001) (Figure 4C). We suggest two possible scenarios that may contribute to this decrease in accuracy. First, the linear ion trap may be less accurate or precise than the Orbitrap in measuring spectral intensity. Second, the higher number of ions and thus improved overall ion statistics in the Orbitrap compared to the linear ion trap, which were analyzed with AGC targets of 5.0e4 and 5.0e3, respectively, may allow for more accurate and precise measurements of ion intensity. It is evident, however, that the small decrease in accuracy and precision when performing the MS3 scan in the linear ion trap is consistently paired with a highly significant increase in overall number of PSM and overall proteins identified through quantification in all six reporter channels (Figure 4, Figure S3, Table S2 and Table S3). On average, due to the multiplexing across six channels, performing the HCD-MS3 scan in the linear ion trap resulted in 13,374 protein-quantification events (i.e. on average, 2,229 proteins quantified per replicate times six reporter channels equals 13,374 protein-quantification events per replicate) in a 2-hour analysis, compared to the 8,052 events observed when the Orbitrap was used (Table S3). Performing the HCD-MS3 scan in the linear ion trap, furthermore, leads to similar numbers of highly accurate (1 peptide than when the Orbitrap HCD-MS3 scan was used (Figure 4C). It should be noted that fractionation of samples prior to mass spectrometry analysis will increase the number of proteins that are identified and quantified. The accuracy and precision from fractionated and unfractionated samples were comparable, in both one and two-proteome models (data not shown). For this study, in which the major goal was to compare protein quantification when the MS3 scan was acquired in an ion trap vs. an Orbitrap, we reasoned that the use of an unfractionated sample was appropriate. These results would also be particularly relevant when instrumentation time is limited. In summary, the use of a linear ion trap for the HCD-MS3 scan while performing a 6-plex TMT experiment allows for both powerful multiplexed quantitative analysis and high number of protein identification events. In this method, the ion

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A

B

C

Figure 4. Comparison of distribution of percent error, compared to the idealized fractional intensity for the combined six reporter channels, based on (A) peptide-spectrum matches (PSM), (B) all proteins identified, and (C) proteins identified by >1 peptide. All data points were quantified in all six reporter channels. Error bars indicate the standard deviation of five technical replicates. ***, p < 1.0e-06; **, p < 1.0 e-04; *, p < 0.01. Italicized values in the figure legends represent the average, across the five replicates, of the median percent error.

trap-HCD MS3 scan takes place after SPS, and thus interference is expected to remain minimized while sensitivity increases. A similar approach cannot be used with 10-plex TMT experiments, as the linear ion trap lacks the resolving power to differentiate between reporter ions that have less than one mass unit resolution (e.g. 127C and 127N) – something that the Orbitrap is able to do. Nonetheless, the use of the ion trap for MS3 scans is expected to positively impact any multiplexed MS3-TMT experiment that uses six channels or fewer. In fact, any set of reporter ions that has at least one mass unit resolution, including iTRAQ 4-plex or 8-plex, and CIT multiplex reagents,15 can be used with the MS3-HCD linear ion trap quantification.

ASSOCIATED CONTENT


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Supporting Information

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REFERENCES

Raw data and search results were uploaded to ProteomeXchange with accession: PXD004093. Supporting Information Available: Extended methods. Figure S1: Summary of data for each of the three AGC targets tested for the MS3-HCD scans when using the linear ion trap for the MS3 scan. Figure S2: Accuracy and precision of peptide quantification when collecting the MS3-HCD scans using the linear ion trap with three different AGC targets. Figure S3: Number of peptide-to-spectrum matches (PSM) that had reporter ions quantified in the indicated amount of reporter channels. Figure S4: Accuracy and precision of protein quantification when collecting the MS3-HCD scan using the linear ion trap or the Orbitrap. Table S1: Quantification data when varying AGC target values in the linear ion trap (MS3-HCD Scan). Table S2: Number of PSM as a function of reporter channels. Table S3: Protein identification rates. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *Email: [email protected]. Tel: (626) 395-2339. Fax: (626) 4494159. *Email: [email protected]. Tel: (909) 607-8832.

Author Contributions The manuscript was written through contributions of all authors.

ACKNOWLEDGMENT This work was supported by Pomona College, the Beckman Institute, and the Gordon and Betty Moore Foundation through Grant GBMF775. We thank Tanya Porras-Yakushi for providing yeast whole cell lysates and are grateful to Annie Moradian and Roxana Eggleston-Rangel for their assistance with obtaining the mass spectra.

(1) Erickson, B. K.; Jedrychowski, M. P.; McAlister, G. C.; Everley, R. A.; Kunz, R.; Gygi, S. P. Anal. Chem. 2015, 87, 1241-1249. (2) Clark, D. J.; Fondrie, W. E.; Liao, Z.; Hanson, P. I.; Fulton, A.; Mao, L.; Yang, A. J. Anal. Chem. 2015, 87, 10462-10469. (3) Viner, R. I.; Zhang, T.; Second, T.; Zabrouskov, V. J. Proteomics 2009, 72, 874-885. (4) Dayon, L.; Hainard, A.; Licker, V.; Turck, N.; Kuhn, K.; Hochstrasser, D. F.; Burkhard, P. R.; Sanchez, J.-C. Anal. Chem. 2008, 80, 2921-2931. (5) 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.; Jacobson, A.; Pappin, D. J. Mol. Cell. Proteomics 2004, 3, 11541169. (6) Thompson, A.; Schäfer, J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.; Neumann, T.; Hamon, C. Anal. Chem. 2003, 75, 18951904. (7) Altelaar, A. F. M.; Frese, C. K.; Preisinger, C.; Hennrich, M. L.; Schram, A. W.; Timmers, H. T. M.; Heck, A. J. R.; Mohammed, S. J. Proteomics 2013, 88, 14-26. (8) Ting, L.; Rad, R.; Gygi, S. P.; Haas, W. Nat. Methods 2011, 8, 937-940. (9) McAlister, G. C.; Nusinow, D. P.; Jedrychowski, M. P.; Wühr, M.; Huttlin, E. L.; Erickson, B. K.; Rad, R.; Haas, W.; Gygi, S. P. Anal. Chem. 2014, 86, 7150-7158. (10) Kalli, A.; Sweredoski, M. J.; Hess, S. Anal. Chem. 2013, 85, 3501-3507. (11) Wiśniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Nat. Methods 2009, 6, 359-362. (12) Manza, L. L.; Stamer, S. L.; Ham, A.-J. L.; Codreanu, S. G.; Liebler, D. C. Proteomics 2005, 5, 1742-1745. (13) Moradian, A.; Porras-Yakushi, T. R.; Sweredoski, M. J.; Hess, S. Methods Mol. Biol. 2016, 1394, 75-85. (14) Kim, S.; Pevzner, P. A. Nat. Commun. 2014, 5, 5277. (15) Sohn, C. H.; Lee, J. E.; Sweredoski, M. J.; Graham, R. L. J.; Smith, G. T.; Hess, S.; Czerwieniec, G.; Loo, J. A.; Deshaies, R. J.; Beauchamp, J. L. J. Am. Chem. Soc. 2012, 134, 2672-2680.



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Figure 3. Accuracy and precision of (A) peptide and (B) protein quantification when collecting the MS3-HCD scan using the linear ion trap (dark blue, left) or the Orbitrap (light blue, right). The proteins analyzed were all identified by >1 peptide. Boxplots indicate the median (middle line), the interquartile range (box) and the 5th and 95th percentile for each experiment (which includes 5 technical replicates). The dashed lines indicate the values for the idealized fractional intensities. 134x73mm (300 x 300 DPI)



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Figure 3. Accuracy and precision of (A) peptide and (B) protein quantification when collecting the MS3-HCD scan using the linear ion trap (dark blue, left) or the Orbitrap (light blue, right). The proteins analyzed were all identified by >1 peptide. Boxplots indicate the median (middle line), the interquartile range (box) and the 5th and 95th percentile for each experiment (which includes 5 technical replicates). The dashed lines indicate the values for the idealized fractional intensities. 134x74mm (300 x 300 DPI)


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Improved 6-Plex Tandem Mass Tags Quantification Throughput Using

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