Liver Mitochondria Proteomics Employing High-Resolution MS Technology

Transcription

1 Liver Mitochondria Proteomics Employing High-Resolution MS Technology Jenny Ho, 1 Loïc Dayon, 2 John Corthésy, 2 Umberto De Marchi, 2 Antonio Núñez, 2 Andreas Wiederkehr, 2 Rosa Viner, 3 Michael Blank, 3 Steve Danielson, 3 Madalina Oppermann, 1 Martin Hornshaw, 1 2, 4, 5 Martin Kussmann 1 Thermo Fisher Scientific, Hemel Hempstead, UK, 2 Nestlé Institute of Health Sciences, Lausanne, Switzerland, 3 Thermo Fisher Scientific, San Jose, USA, 4 Faculty of Life Sciences, Ecole Polytechnique Fédérale Lausanne (EPFL), Lausanne, Switzerland, 5 Faculty of Sciences, Aarhus University, Aarhus, Denmark

2 Overview Purpose: Combine label-free and TMT-based relative quantification for deep differential profiling of the liver mitochondrial proteome. Methods: Complementary analytical workflows combining nanolc with highresolution, accurate-mass measurements using the Thermo Scientific Orbitrap Elite mass spectrometer. Results: Approximately 1,500 protein groups were identified at 1% FDR, where more than 50% of these were mitochondrial proteins. Relative abundances of mitochondrial proteins determined by label free and TMT-labeling methods, respectively, were consistent. Introduction Mitochondrial dysfunction has been linked to chronic metabolic disorders such as type 2 diabetes and neurodegenerative diseases, while accumulation of mitochondrial damage has been associated with the process of aging. To develop methodology for use in human samples, mouse liver mitochondria were isolated and purified in the presence of protease inhibitors. The protein fraction was digested with trypsin and analyzed by complementary nanolc-ms/ms workflows to maximize both qualitative and quantitative information on the mitochondrial proteome, phosphoproteome and acetylome. Methods Sample Preparation Tissues were homogenized in control buffer (non-treated) or buffer containing phosphatase inhibitors (treated). Mitochondria were isolated by sub-cellular fractionation and Percoll gradient centrifugation [1]. Proteins were precipitated with methanol/ chloroform/water. Proteins were re-dissolved, cysteines reduced and alkylated, and proteins were digested with trypsin. The digested samples were split into three and used for (a) global label-free quantitation, (b) TMT-labeling quantitation and (c) phosphopeptide enrichment (Figure 1). Figure 1. Workflow combining relative quantitation and phosphopeptide enrichment to maximize mitochondrial proteome. 2 Liver Mitochondria Proteomics Employing High-Resolution MS Technology

3 LC-MS/MS All samples were analyzed by nanolc coupled to the Orbitrap Elite mass spectrometer. Peptides were separated using a Thermo Scientific Acclaim PepMap C18 column, 500mm x 75µm ID, 2µm, employing a water/acetonitrile/0.1% formic acid gradient from 5-30% over 205 minutes. For global label-free quantitation, peptides were analyzed by data-dependent acquisition, where full MS spectra were acquired at 120,000 resolving power and the 20 most intense multiply-charged precursors were selected for ion trap CID. 1µg of material was loaded on-column and samples were analyzed in triplicate. For TMT quantitation, peptides were analyzed by data-dependent-acquisition, where full MS spectra were acquired at 120,000 resolving power and the 15 most intense multiplycharged precursors were selected for HCD (higher energy collisional dissociation). Fragment ions were measured at 30,000 resolving power. For enriched phosphopeptide samples, peptides were analyzed by data-dependent Top15 MSA (multi-stage activation), where full MS spectra were acquired at 120,000 resolving power and the 15 most intense multiply-charged precursors were selected for MSA. Product ions were measured in the ion trap. Data Analysis For peptide and protein identification as well as TMT quantitation, data were processed using Thermo Scientific Proteome Discoverer 1.3 software. Spectra were searched against a Uniprot mouse database using the Mascot (Matrix Science) or SEQUEST search engines. Label-free quantitation was performed using Thermo Scientific SIEVE 2.0 software. Thermo Scientific Poster Note PN64036_HUPO2014 _E 04/14S 3

4 Results Label Free Approach Trypsin digests were analyzed in triplicate using a 205 minute gradient. Base peak chromatograms for non-treated and treated samples are shown in figure 2. Figure 2. (A) Base peak chromatograms and (B) Proteome Discoverer workflow for peptide and protein identification. (A) (B) Database: UnitProt MOUSE. (Dec 2012) Trypsin Cleavage: Up to 2 Missed Cleavages Tolerances: Precursor 10ppm, fragment ion 0.6Da Dynamic Modifications: Acetyl (N-term, K, Phospho (S, T, Y), Oxidation (M) Static Modifications: Carbamidomethylation (C) 4 Liver Mitochondria Proteomics Employing High-Resolution MS Technology

5 Approximately 1,400 protein groups were identified at 1% FDR (combining three replicate experiments). Gene ontology annotation indicated that over 50% of the protein groups identified were mitochondrial, representing a high number of specific identities for organelle-based enrichment. Figure 3 shows a bar chart summarizing the number of peptides and proteins identified and quantified by label-free analysis and a pie chart showing the distribution of mitochondrial proteins identified. 36 phosphopeptides and 63 acetyllysine containing peptides were confidently identified without prior PTM enrichment (5µg load, data-dependent Top10HCD, 420min gradient). Figure 3. (A) Bar chart summarizing the number of proteins and peptide spectrum matches (PSM) identified and quantified by label free quantitation, and (B) pie chart showing distribution of identified mitochondrial protein groups (n=3). (A) (B) Other proteins Mitochondrial Data files from non-treated (n=3) and treated (n=3) were processed using SIEVE 2.0 software. Briefly, LC chromatograms were aligned and peaks were detected. The volcano plot shown in Figure 4 highlights the summary of protein ratios (log2) versus pvalue. 94% of the proteins identified were quantified. Figure 4. Volcano plot of protein ratios vs pvalues. TMT-labeling An aliquot of the non-treated and treated trypsin digested samples were labeled with Thermo Scientific TMT6plex labeling reagents (three TMT tags were used to label non-treated and three TMT tags were used to label treated samples) following the manufacturer s protocol. The labeled samples were mixed 1:1:1:1:1:1 and analyzed by nanolc-ms/ms. Peptides were analyzed by data-dependent Top15 HCD, where fragment ions were measured at 30,000 resolution at m/z 400, resulting in an effective resolution of over 56,000 for TMT reporter ions with their masses around m/z 130. At this resolving power, complete separation of A+1(2)/A-1(2) isotopes was achieved, which allowed a mass tolerance of +/-10ppm for reporter ion detection, thereby eliminating impurities and ensuring more accurate quantitation. Reporter ions were measured with less than 1 ppm error in the example shown in Figure 5(A) of an identified TMT-labeled peptide. Thermo Scientific Poster Note PN64036_HUPO2014 _E 04/14S 5

6 Figure 5. (A) Example of annotated spectrum of a TMT-labeled peptide and mass accuracy of reporter ions and (B) Proteome Discoverer workflow for peptide/protein identification and quantitation. (A) (B) Database: UnitProt MOUSE. (Dec 2012) Trypsin Cleavage: Up to 2 Missed Cleavages Tolerances: Precursor 10ppm, fragment ion 0.02Da Dynamic Modifications:Deamidation (NQ), Oxidation (M), TMT6 Static Modifications: Carbamidomethylation (C) 6 Liver Mitochondria Proteomics Employing High-Resolution MS Technology

7 Approximately 1,300 protein groups were identified at 1% FDR (combining n=3). Figure 6 shows a bar chart summarizing the number of peptides and proteins identified and quantified and a pie chart showing the distribution of identified mitochondrial proteins. 98% of the identified proteins were quantified. Figure 6: (A) Bar chart summarizing the number of peptides and proteins quantified by TMT and (B) pie chart showing the distribution of identified mitochondrial proteins. (A) (B) TMT-labeling Other Mitochondrial proteins Other Mitochondrial Proteins Although a similar gradient length was used for both the label-free analysis employing data-dependent Top20CID, a slightly lower number of unique peptides (~15%) was observed by data-dependent Top15 HCD employed for TMT quantitation. This is likely due to MS/MS sampling time and narrow isolation width (1.2 amu vs. 2 amu). The number of proteins groups identified by the label-free approach was comparable to the TMT-labeling (increase of ~10%), however this took significantly longer, almost double the analysis time. 98% of the proteins identified by TMT-labeling were quantified compared to 94% using the label-free approach. Quantitative Precision and Biological Significance Relative abundances of mitochondrial proteins determined by label-free and TMTbased methods were highly conserved. Table 1 shows examples of protein ratios determined by label-free and TMT quantitation. Thermo Scientific Poster Note PN64036_HUPO2014 _E 04/14S 7

8 Table 1. Examples of proteins identified and quantified by both labelfree and TMT-labeling methods. 8 Liver Mitochondria Proteomics Employing High-Resolution MS Technology

9 Using TMT-labeling, 93% of the identified proteins were quantified with 20% CV (for replicate analyses, the protein ratio CV is calculated from protein ratios in individual replicate runs), whilst 83% of the identified proteins were quantified with 20% CV based on LC peaks detected in each data file using the label free approach (Figure 7). Figure 7. Label-free and TMT-labeling analytical quantitative precision. Several proteins from the Oxidative Phosphorylation Regulation pathway were identified and quantified. Figure 8 shows protein ratios quantified by label-free and TMT-labeling of selected proteins identified in the pathway. Coverage of the protein members of this pathway displays similar quantitative trends, regardless of the relative quantitation approach used. Figure 8. Quantitation profiles of proteins in the oxidative phosphorylation pathway. Thermo Scientific Poster Note PN64036_HUPO2014 _E 04/14S 9

10 Phosphopeptide Analysis Phosphopeptide enriched samples were analyzed using data-dependent Top15MSA. More than 250 unique phosphopeptides were identified at 1% FDR with a PhosphoRS score 50 employing the PhosphoRS node in Proteome Discoverer software v1.3. Label-free quantitation was performed post-processing using Thermo Scientific Pinpoint software, where peak areas from the three most abundant isotopologues of the precursor were used (Figure 9A). Differences in relative abundance levels were observed between phosphopeptides quantified in the non-treated and phosphatase inhibitors treatment, as expected. 63% of the quantified phosphopeptides showed higher abundances in samples treated with phosphatase inhibitors while 35% showed no change suggesting that the majority of the phosphate groups were protected from phosphatase activity. Duplicate injections of each sample were performed and the extracted ion chromatograms show good reproducibility. Examples of identified and quantified phosphopeptides are shown in Figure 9B. A higher number of modified sites were identified and quantified than previously reported [2], which is of relevance towards the understanding of biological functions associated with the mitochondrion. 10 Liver Mitochondria Proteomics Employing High-Resolution MS Technology

11 Figure 9. Examples of quantified phosphopeptides (A) extracted ion chromatograms from Pinpoint software and (B) phosphopeptides observed to be higher, lower and no change in abundances in the treated samples compared to the non-treated samples. (A) (B) Thermo Scientific Poster Note PN64036_HUPO2014 _E 04/14S 11

12 Conclusion The multiplexing capabilities of TMT yielded a similar number of identified and quantified proteins to label-free approach, with an equivalent statistical confidence, but for a fraction (1/6) of analysis time. Relative abundances of mitochondrial proteins determined by label-free and TMT-labeling methods, respectively, were comparable. Enrichment using TiO 2 significantly increased the number of identified phosphopeptides. Relative ratios for 288 phosphopeptides (including missed cleavages and methionine oxidiation) were calculated using extracted ion chromatograms (XIC) from the full MS spectra. Differences in the relative abundance levels of phosphopeptides were observed between control and phosphatase inhibitor treatment conditions, this is likely due to the protection of phosphate groups from phosphatase activity. References [1] Sims et al. (2008) Nature Protocols, 3, [2] Gnad et al. (2010), Mol Cell Proteomics, 9 (12): Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. SEQUEST is a trademark of the University of Washington. All other trademarks are the property of Thermo Fisher Scientific, Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Africa Australia Austria Belgium Canada China (free call domestic) Denmark Europe-Other Finland France Germany India Italy Japan Latin America Middle East Netherlands New Zealand Norway Russia/CIS Thermo Fisher Scientific, San Jose, CA USA is ISO 9001:2008 Certified. Singapore Spain Sweden Switzerland UK USA HUPO2014_PN64036_E 04/14S