3c). 6), we used a 90-min gradient at a flow rate of 250nl/min ramping from 10% buffer B (80% ACN and 0.1% formic acid) to 30% B in 69min, to 45% B in 13min, to 80% B in 2min, kept 2min, to 5% B in 2min and kept 2min. Comparing SILAC- and stable isotope dimethyl-labeling approaches for quantitative proteomics. helped set up the microflow high pH fractionation system. We used an R package provided by Tibshirani et al.45, which can automatically estimate optimal s0 based on the tested data, and calculates a d-score representing the degree of significant regulation of each tested phosphopeptide. 86, 35943601 (2014). 3e). This s0 parameter gives greater weight to extreme fold changes and should be adjusted to the data set at hand44. The poor performance of LFQ was not simply due to the broader phosphorylation site coverage of TMT. In addition, the increase in accuracy of MS3-based over MS2-based for TMT quantification is essentially negated by its even bigger decrease in precision. b Heatmap of correlation slopes of the 5% highest and lowest log2 ratios for all replicates. SILAC-Based Quantitative Phosphoproteomics in Yeast A.H. designed and performed all experiments, and analyzed the data and wrote the paper. 75, 18951904 (2003). Cell 127, 635648 (2006). Biotechnol. SILAC-labeled peptides from digested whole cell lysates are as a first step enriched for phosphorylated tyrosines by immunoaffinity and then further enriched for phosphorylated serine/threonine peptides by strong cation exchange in combination with titanium dioxide-beads chromatography. Research TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics* Jun Zhong , Min-Sik Kim , Raghothama Chaerkady , Xinyan Wu , Tai-Chung Huang , Derese Getnet , Christopher J. Mitchell , Shyam M. Palapetta ** , Jyoti Sharma , Robert N. O'Meally , Robert N. Cole , Akinori Yoda , Albrecht Moritz , 13, 61876195 (2014). Article To achieve reliable biological interpretation, quantification methods have to be both accurate and precise. Human epithelial cervix carcinoma HeLa cells (female) and human epithelial osteosarcoma U2OS cells (female) were purchased from ATCC. Three of them used multi-stage activation (MSA)-combined low energy collisional-induced dissociation (CID) and synchronous precursor selection (SPS) of the ten most abundant MS2 fragment ions33. After concluding that the four quantification methods can identify different numbers of SAM-regulated phosphorylation sites, we wanted to assess if and to which degree these sites gave us biological insight into the cellular signaling of the doxorubicin-induced DDR. Evaluation of quantification methods with focus on accuracy and precision. For the gradient experiment (bd), samples were measured in three biological replicates using a 15-cm column with a 30, 90 or 180-min gradient, or a 50-cm column with a 290-min gradient on a Q Exactive HF-X. Stable isotope labeling by amino acids in cell culture (SILAC) has long been the preferred method for phosphopeptide quantification1,18, but recently label-free quantification (LFQ)2,19,20 and isobaric tandem mass tags (iTRAQ, TMT)3 have become popular for phosphoproteomics. For this purpose, we used the well-studied DDR as a model system (Fig. PubMed Central For phosphorylation site analysis, the lowest available underscore intensity entries from the MaxQuant output were used. In addition, SILAC and otherlabeling approaches with MS1-based quantification have inherently higher MS1 spectral complexity. Phosphopeptides are then eluted with 5% ammonia (Merck, 1054321011) and 10% ammonia/25% ACN and subsequently loaded onto C18 StageTips (made from Sigma-Aldrich, 66883-U). Furthermore for the technical benchmarks, SILAC incorporation efficiency was corrected by using missed cleavage-separated correlation factors between MaxQuant raw and normalized SILAC ratio columns, extracted from a 1:1:1 mixture of HeLa or yeast, respectively. Huang et al. SILAC is impacted by this as well, when requiring quantification in more than one biological replicate. Acad. MATH Three biological replicates were measured for all quantification methods. This can be done either relatively by calculation of ratios between conditions, or absolute within conditions, often referred to as phosphorylation site stoichiometry or occupancy15,16,17. Chem. Cells were treated with genotoxic agents doxorubicin (DOX), 4-nitroquinoline 1-oxide (4NQO) or DMSO as a control (C) in biological triplicates for 2h before lysis. The NL-triggered method showed a better decompression of 1.72, but at the same time generated the lowest number of phosphopeptide ratios of all five methods (Fig. Twenty-four concatenated fractions consisting of pooled fractions of variable time length were collected and directly subjected to MS measurement. It is additionally complicating that the number of identified yeast and human peptides vary between the quantification approaches due to the inherent stochastic behavior of the data-dependent acquisition (DDA). For SILAC and TMT, MS samples were injected without dilution, so that each labeling channel resembles one LFQ injection. 6 is available as an R script in the supplementary section as Supplementary Data1 and example data from Fig. For SILAC and TMT, MS samples were diluted to contain a total peptide amount equal to one LFQ injection based on protein starting amount. PubMed Introduction Phosphorylation is one of the commonest posttranslational modifications involved in regulating biological processes in a cell. Cell. DNA was sheared by 2-min ultrasonication treatment (Sonics & Materials, VCX 130; 1s on, 1s off, 80% amplitude). Sci. 3). PubMed d Receiver operating characteristic (ROC) curves were calculated by using the d-score from SAM testing as an indicator for significant regulation at 4:1 and 10:1 dilution. SILAC removes false positives in protein-interaction studies, reveals large-scale kinetics of proteomes and as a quantitative phosphoproteomics technology directly uncovers important points. Boxes mark the first and third quartile, with the median highlighted as dash, and whiskers marking the minimum/maximum value within 1.5 interquartile range. 2). Linear sequence motif analysis of the upregulated phosphorylation sites revealed that all techniques, including MS2-based TMT, could correctly identify the DDR-induced ATM/ATR kinase substrate motif [s/t]Q as significantly enriched (Fig. The resulting over-sequencing of peptide isotope-variants leads to a decrease in total peptide identifications22,23. However, this disadvantage might be counterbalanced by activating MBR or measuring more replicates instead. 4c,d). Savitski, M. M. et al. SILAC-based phosphoproteomics reveals an inhibitory role of - Nature SILAC-based phosphoproteomics reveals new PP2A-Cdc55-regulated processes in budding yeast | GigaScience | Oxford Academic AbstractBackground. 3), we used a 70-min gradient ramping from 10% B to 30% B in 54min, to 45% B in 10min, to 80% B in 1min, kept 2min, to 5% B in 1min and kept 2min. Nature Communications & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Since the true target ratios were known, we were able to calculate the fraction of reporter ion intensity coming from contaminating ions for MS2-based TMT. Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response. Article 1355, 251260 (2016). 6c). In contrast, MS3-based TMT quantification-derived stoichiometry is highly accurate even without any p-value cutoffs. Samples were then measured using a 15- or 50-cm (only LFQ) column on a Q Exactive HF or Orbitrap Fusion Lumos (only TMT MS3 OT MC). Proteom. 3), four replicates were measured, but only three were used at random in the data analysis, equal to SILAC and TMT. ggplot2: Elegant Graphics for Data Analysis. Cell. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. With recent developments in LFQ-based data independent acquisition (DIA) quantification46, it might be interesting to see how this approach can compare to TMT multiplexing for quantitative phosphoproteomics experiments, once current DIA limitations such as reliable phosphorylation site localization can be routinely addressed. Phosphoproteomes of SILAC-labeled mouse embryonic stem cells (mESCs) were generated after exposure to ionizing radiation . A tissue-specific atlas of mouse protein phosphorylation and expression. We would like to stress that this analysis of course depends on many factors, including chosen ratios, total MS ion intensities and the applied statistical test. We found that it negatively correlated with MS1 precursor intensity, Andromeda MS/MS score and precursor isolation fraction (PIF) (Supplementary Fig. TMT correction factors in MaxQuant were updated to the values provided by the manufacturer. Online ahead of print. doi: 10.1021/acs.jproteome.1c00735. Batth, T. S., Francavilla, C. & Olsen, J. V. Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics. 3), we used a 290-min gradient ramping from 5% B to 30% B in 240min, to 80% B in 35min, kept 5min, to 5% B in 5min and kept 5min. As expected, all MS3-based TMT methods were able to decompress the observed MS2-based TMT ratios, but to different extents (Fig. Article usually towards unity30, but also higher apparentprecision31. 1b, Supplementary Figs. Beads were transferred to C8 StageTips (made from Sigma Aldrich, 66882-U)50 and washed with 10% ACN/6% TFA, 40% ACN/6% TFA, and 60%/6% TFA. In this analysis, we aimed at evaluating different MS quantification methods both from a technical perspective, as well as with a biological focus on identification of significantly regulated phosphopeptides. 5). Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates. ROC plots are presented as zoomed-in excerpts from the total plots, shown on the lower right each. 2d). Natl. Comparing the basal phosphoproteome of cell lines AU565 vs. T47D in technical duplicates confirms that while MS3-based TMT decompresses MS2-measured ratios, the latter allows the identification of more significantly regulated phosphorylation sites (Supplementary Fig. When measuring these samples in both MS2- and MS3-mode, we found that we can assess the quality of the 3DMM linear fit by calculating a p-value, which describes the significance of the slope being non-zero. ADS The latter is caused by ratio compression via impure MS1 precursor isolation29. We further noted that all MS3-based analyses had a significantly lower number of phosphopeptide ratios and lower mediansignal-to-noise ratios of the TMT reporter ions (Fig. Phosphorylation stoichiometry can be extracted directly from large-scale quantitative phosphoproteomics experiments by using ratios observed in both the phosphopeptide, its non-phosphorylated counterpart and the respective protein between treatment conditions15, or directly from the phosphopeptide within a single treatment condition16,17. Chem. D.B.B.-J. In addition, we found that p-values extracted from the fit of our 3DMM can estimate the quality and serve as cutoff values, which is especially useful for MS2-based TMT quantification. MBR is now widely employed in LFQ- and SILAC-based studies and similar concepts are implemented in many proteomics software tools. What is Phosphoproteomics? - News-Medical.net Cell 143, 11741189 (2010). We thus believe it highlights the potential of linear modeling-based TMT data analysis, not only for phosphorylation stoichiometry, but for other PTM applications as well. Day cultures were inoculated at OD600 of ~0.1 and harvested at OD600 of ~0.9. Work at The Novo Nordisk Foundation Center for Protein Research (CPR) is funded in part by a generous donation from the Novo Nordisk Foundation (grant number NNF14CC0001). Nature 534, 5562 (2016). Introduction and summary of different methods of protein quantification, including label-free and labeling techniques used in proteomics approach applicable in cancer research, such as stable-isotope labelling by amino acids in cell culture (SILAC), tandem mass tags (TMT), and Isobaric tag for relative and absolute quantitation (iTRAQ) Giai Gianetto, Q., Cout, Y., Bruley, C. & Burger, T. Uses and misuses of the fudge factor in quantitative discovery proteomics. Science 316, 11601166 (2007). (2017)47 were downloaded from MassIVE with identifier MSV000079655 (ftp://massive.ucsd.edu/MSV000079655). Nevertheless, we can conclude that, especially for the biologically more interesting 10:1 ratios, LFQ, SILAC, MS3 OT MC-based and even ratio-compressed MS2-based TMT, all seem to provide good and comparable compromises between sensitivity and specificity. Briefly, the FDR was set to 1% on peptide spectrum match (PSM), PTM site and Protein level. 3, 16301638 (2008). A. et al. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. You are using a browser version with limited support for CSS. Vizcano, J. & Li, J. samr: SAM: Significance Analysis of Microarrays. 32, 223226 (2014). c 3DMM-extracted p-values describing the significance of the slope being non-zero were correlated against the difference of MS2- and MS3-estimated stoichiometry vs. the true value of 10%. Signal. 2and Supplementary Fig. Nat. Accurate multiplexed proteomics at the MS2 level using the complement reporter ion cluster. 2), were quantile normalized using the R package preprocessCore v1.40.054. Quantitative Phosphoproteomics Reveals Pathways for Coordination of McAlister, G. C. et al. SILAC phosphoproteomics reveals unique signaling circuits in - bioRxiv Proteomics 16, 907914 (2016). Next we assessed how the different tradeoffs in accuracy and precision for the quantification approaches would influence their ability to identify phosphopeptides as significantly regulated. Chem. However, even though we did not test this with our setup, MS3-based TMT multiplexing over 11 treatment conditions, instead of three in SILAC, makes it more likely that a biological change is observed, and further increases precision of the modeling. After rocking for 5min, cells were scraped and lysate was boiled for 10min at 95C. Nat. However, SILAC is limited to cell lines and a maximum of three conditions per sample in routine analysis. Stable heavy isotope-labeled or unlabeled amino acids are incorporated metabolically into cells, and differentially labeled cell populations can be mixed directly after lysis21. PubMed 3b). Yeast pellet powder was supplemented with 1% Triton X-100 (Sigma-Aldrich, T9284) v/v and thawed at 4C for 30-min rolling. Even when analyzing more than 11 conditions, where current-generation TMT would start suffering from missing values as well, this issue would be even more severe for LFQ and SILAC. 11, M111 014423 (2012). 13, 61766186 (2014). Peptides were separated by a linear gradient from 5% B to 25% B in 50min followed by a linear increase to 75% B in 5min, and kept there for 5min before ramping to 5% in 5min. We would like to thank the PRO-MS Danish National Mass Spectrometry Platform for Functional Proteomics and the CPR Mass Spectrometry Platform for instrument support and assistance. All raw files were then processed by the MaxQuant software suite (www.maxquant.org) for identification and quantification13. All raw MS data files from this study have been deposited to the ProteomXchange Consortium59 via the PRIDE partner repository60 with identifier PXD007145 (https://www.ebi.ac.uk/pride/archive/projects/PXD007145). As a tradeoff compared to LFQ, isobaric quantification approaches usually yield decreased peptide identificationrates22,28 and lowered accuracy when quantified on MS2-level23. Interestingly, we found that the higher accuracy of SPS-MS3-based TMT approaches is directly negated by its lowered precision. We would expect that if the gain in precision is indeed caused solely by such a quantification artifact, MS2-based TMT should perform much worse than MS3-based TMT at deeming phosphopeptides significantly regulated. All samples were measured on an Orbitrap Fusion Lumos three times as technical replicates with each quantification method. 15, 46534665 (2016). Peptides were eluted with 40 and 60% ACN and subjected to TMT labeling or directly to MS measurement. 86, 71507158 (2014). Werner, T. et al. This way we can assess how the different methods would quantify the expected ratios (Fig. Total proteomics (protein expression data) and phosphoproteomics experiments were processed separately. Cox, J. et al. Phosphoproteomics - Wikipedia Our input data sets were sequence windows for SAM upregulated phosphorylation sites of each quantification method. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Protoc. Proteom. This might be influenced by the higher scan speed and brighter ion source of the HF-X, which could lead to higher ion intensities and thus better statistics, even though we have not tested this in a direct comparison46. We applied it by adapting stoichiometry estimation to TMT-based quantification, using a newly developed 3D multiple regression model-based approach, which takes advantage of the high multiplexing capabilities of TMT. a iceLogos of the SAM-upregulated phosphorylation sites from Fig. As expected, MS2-based TMT ratios were heavily affected by ratio compression, resulting in the lowest accuracy of all compared quantification methods. In the meantime, we advise large-scale phosphoproteomics users to consider using MS2-based TMT-based quantification, as long as reproducible but not necessarily accurate quantification is required. We performed dilution experiments and determined that 1l TMT reagent is sufficient to label phosphopeptides equivalent to ~1mg protein starting material to reach a labeling efficiency of >95% for the cell lines used in this publication. Bekker-Jensen, D. B. et al. High-performance hybrid Orbitrap mass spectrometers for quantitative proteome analysis: observations and implications. We found that TMT-labeling seemed to make the peptides more hydrophobic and tried to optimize our gradients based on this observation. For this purpose, we developed a 3D multiple regression model (3DMM)-approach that makes use of the high TMT multiplexing capabilities. SAM testing for significantly regulated phosphopeptides was performed at default settings (s0 estimation automatic). Wiese, S., Reidegeld, K. A., Meyer, H. E. & Warscheid, B. We then identified significantly regulated phosphorylation site ratios among the remaining sites via SAM testing (Fig. Google Scholar. To test this, we repeated the experiment only for LFQ with DOX treatment, but this time varied the gradient length to include 30, 90 and 180min on a 15-cm column, and our original 290-min 50-cm approach (Fig. The column temperature was maintained at 40C or 50C for the 15-cm and 50-cm column, respectively, using an integrated column oven (PRSO-V1, Sonation). Nat. 2b, Supplementary Fig. Buffer C was constantly added to the gradient at 10%. Comprehensive mass spectrometry (MS)-based proteomics is now feasible, but reproducible quantification remains challenging, especially for post-translational modifications such as phosphorylation. contributed to the experimental design on the D.D.R. Anal. Variable modifications considered were oxidation of methionine, protein N-terminal acetylation, and phosphorylation of serine, threonine and tyrosine residues. 1, 376386 (2002). Olsen, J. V. et al. BY4742 wt yeast cells were grown in yeast medium (drop out mix without lysine, Nordic Biosite, D9515B; 6.7g/l yeast synthetic drop-out medium supplements without lysine, Sigma-Aldrich, Y137620G; 2% v/v glucose, Sigma-Aldrich, G70211KG; 205l/l SILAC lysine 0/4/8) at 30C and 200rpm rotation in overnight cultures. Prior to phosphopeptide enrichment, SILAC samples were fractionated according to a modified protocol by Batth et al.11. Wenger, C. D. et al. Nat. Thus, after confirming that MS3-based TMT methods can decompress MS2-measured phosphopeptide ratios to different extents, we next compared the quantification accuracy and precision of these approaches to that of LFQ and SILAC. For processing of SILAC data, Thermo files (.raw format) were loaded in to MaxQuant (version 1.6.0.16). Mol. These strict requirements exclude most falsely quantified or localized sites, which is commonly done in cell signaling studies. 6d). Interestingly, SILAC yields the highest number of phosphorylation site identifications without quantitative information, but this can to some extent be rescued by activating the REQ feature. LFQ, LFQ-MBR and SILAC-MBR-REQ show the lowest precision of all quantification methods. CAS SILAC phosphoproteomics reveals unique signaling circuits in CAR-T cells and the inhibition of B cell-activating phosphorylation in target cells Alijah A. Griffith , View ORCID Profile Kenneth P. Callahan , View ORCID Profile Nathan Gordo King , View ORCID Profile Qian Xiao , View ORCID Profile Xiaolei Su , Arthur R. Salomon Both TMT-approachesand SILAC still outperformed LFQ when only sites quantified in all eight quantification approaches were used for kinase motif enrichment (Supplementary Fig. e Mean squared errors were calculated as a sum of positive bias and variance for all replicates of both MS2- and MS3-based TMTat different 3DMM p-value cutoffs. Article Proteom. Evaluation of quantification methods in a biological setting. SILAC Phosphoproteomics Reveals Unique Signaling Circuits in CAR-T Cells and the Inhibition of B Cell-Activating Phosphorylation in Target Cells J Proteome Res. SILAC was almost as accurate as LFQ, but underestimated ratios on median. We then show that this p-value, which can be calculated for each 3DMM individually, is a reliable determinant of stoichiometry accuracy (Fig. Even though the latter shows the best compromise between accuracy and precision, it suffers from lower phosphopeptide coverage and low multiplexing capabilities. In this study, we show that the highest accuracy alone does not automatically guarantee the best performance in cell signaling studies.