15N metabolic labeling-TMT multiplexing approach to facilitate the quantitation of glycopeptides derived from cell lines.

The study of glycoproteomics presents a set of unique challenges, primarily due to the low abundance of glycopeptides and their intricate heterogeneity, which is specific to each site. Glycoproteins play a crucial role in numerous biological functions, including cell signaling, adhesion, and intercellular communication, and are increasingly recognized as vital markers in the diagnosis and study of various diseases. Consequently, a quantitative approach to glycopeptide research is essential. One effective strategy to address this need is the use of multiplex glycopeptide labeling. By harnessing the synergies of 15N metabolic labeling via the isotopic detection of amino sugars with glutamine (IDAWG) technique for glycan parts and tandem mass tag (TMT)pro labeling for peptide backbones, we have developed a method that allows for the accurate quantification and comparison of multiple samples simultaneously. The adoption of the liquid chromatography-synchronous precursor selection (LC-SPS-MS3) technique minimizes fragmentation interference, enhancing data reliability, as shown by a 97% TMT labeling efficiency. This method allows for detailed, high-throughput analysis of 32 diverse samples from 231BR cell lines, using both 14N and 15N glycopeptides at a 1:1 ratio. A key component of our methodology was the precise correction for isotope and TMTpro distortions, significantly improving quantification accuracy to less than 5% distortion. This breakthrough enhances the efficiency and accuracy of glycoproteomic studies, increasing our understanding of glycoproteins in health and disease. Its applicability to various cancer cell types sets a new standard in quantitative glycoproteomics, enabling deeper investigation into glycopeptide profiles.

Real-Time Search-Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics.

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed scMS requires high ion injection times and high-resolution spectra to quantify the single-cell signal; however, the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time-intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and present a new acquisition strategy termed RETICLE (RTS enhanced quant of single cell spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. We show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at an MS2 injection time of 750 ms using a 2 h gradient.

Enhancing Proteome Coverage by Using Strong Anion-Exchange in Tandem with Basic-pH Reversed-Phase Chromatography for Sample Multiplexing-Based Proteomics.

Sample multiplexing-based proteomic strategies rely on fractionation to improve proteome coverage. Tandem mass tag (TMT) experiments, for example, can currently accommodate up to 18 samples with proteins spanning several orders of magnitude, thus necessitating fractionation to achieve reasonable proteome coverage. Here, we present a simple yet effective peptide fractionation strategy that partitions a pooled TMT sample with a two-step elution using a strong anion-exchange (SAX) spin column prior to gradient-based basic pH reversed-phase (BPRP) fractionation. We highlight our strategy with a TMTpro18-plex experiment using nine diverse human cell lines in biological duplicate. We collected three data sets, one using only BPRP fractionation and two others of each SAX-partition followed by BPRP. The three data sets quantified a similar number of proteins and peptides, and the data highlight noticeable differences in the distribution of peptide charge and isoelectric point between the SAX partitions. The combined SAX partition data set contributed 10% more proteins and 20% more unique peptides that were not quantified by BPRP fractionation alone. In addition to this improved fractionation strategy, we provide an online resource of relative abundance profiles for over 11,000 proteins across the nine human cell lines, as well as two additional experiments using ovarian and pancreatic cancer cell lines.

Proteome Landscape during Ripening of Solid Endosperm from Two Different Coconut Cultivars Reveals Contrasting Carbohydrate and Fatty Acid Metabolic Pathway Modulation.

Cocos nucifera L. is a crop grown in the humid tropics. It is grouped into two classes of varieties: dwarf and tall; regardless of the variety, the endosperm of the coconut accumulates carbohydrates in the early stages of maturation and fatty acids in the later stages, although the biochemical factors that determine such behavior remain unknown. We used tandem mass tagging with synchronous precursor selection (TMT-SPS-MS3) to analyze the proteomes of solid endosperms from Yucatan green dwarf (YGD) and Mexican pacific tall (MPT) coconut cultivars. The analysis was conducted at immature, intermediate, and mature development stages to better understand the regulation of carbohydrate and lipid metabolisms. Proteomic analyses showed 244 proteins in YGD and 347 in MPT; from these, 155 proteins were shared between both cultivars. Furthermore, the proteomes related to glycolysis, photosynthesis, and gluconeogenesis, and those associated with the biosynthesis and elongation of fatty acids, were up-accumulated in the solid endosperm of MPT, while in YGD, they were down-accumulated. These results support that carbohydrate and fatty acid metabolisms differ among the developmental stages of the solid endosperm and between the dwarf and tall cultivars. This is the first proteomics study comparing different stages of maturity in two contrasting coconut cultivars and may help in understanding the maturity process in other palms.

Assessing interference in isobaric tag-based sample multiplexing using an 18-plex interference standard.

Reporter ion interference remains a limitation of isobaric tag-based sample multiplexing. Advances in instrumentation and data acquisition modes, such as the recently developed real-time database search (RTS), can reduce interference. However, interference persists as does the need to benchmark upstream sample preparation and data acquisition strategies. Here, we present an updated Triple yeast KnockOut (TKO) standard as well as corresponding upgrades to the TKO viewing tool (TVT2.5, http://tko.hms.harvard.edu/). Specifically, we expand the TKO standard to incorporate the TMTpro18-plex reagents (TKO18). We also construct a variant thereof which has been digested only with LysC (TKO18L). We compare proteome coverage and interference levels of TKO18 and TKO18L data that are acquired under different data acquisition modes and analyzed using TVT2.5. Our data illustrate that RTS reduces interference while improving proteome coverage and suggest that digesting with LysC alone only modestly reduces interference, albeit at the expense of proteome depth. Collectively, the two new TKO standards coupled with the updated TVT represent a convenient and versatile platform for assessing and developing methods to reduce interference in isobaric tag-based experiments.

Categorization of Phosphorylation Site Behavior during the Diauxic Shift in Saccharomyces cerevisiae.

Protein phosphorylation has long been recognized as an essential regulator of protein activity, structure, complex formation, and subcellular localization among other cellular mechanisms. However, interpretation of the changes in protein phosphorylation is difficult. To address this difficulty, we measured protein and phosphorylation site changes across 11 points of a time course and developed a method for categorizing phosphorylation site behavior relative to protein level changes using the diauxic shift in yeast as a model and TMT11 sample multiplexing. We classified quantified proteins into behavioral categories that reflected differences in kinase activity, protein complex structure, and growth and metabolic pathway regulation across different phases of the diauxic shift. These data also provide a valuable resource for the study of fermentative versus respiratory growth and set a new benchmark for temporal quantitative proteomics and phosphoproteomics for the diauxic shift in Saccharomyces cerevisiae. Data are available via ProteomeXchange with identifier PXD022741.

Temporal Proteomic Profiling of SH-SY5Y Differentiation with Retinoic Acid Using FAIMS and Real-Time Searching.

The SH-SY5Y cell line is often used as a surrogate for neurons in cell-based studies. This cell line is frequently differentiated with all-trans retinoic acid (ATRA) over a 7-day period, which confers neuron-like properties to the cells. However, no analysis of proteome remodeling has followed the progress of this transition. Here, we quantitatively profiled over 9400 proteins across a 7-day treatment with retinoic acid using state-of-the-art mass spectrometry-based proteomics technologies, including FAIMS, real-time database searching, and TMTpro16 sample multiplexing. Gene ontology analysis revealed that categories with the highest increases in protein abundance were related to the plasma membrane/extracellular space. To showcase our data set, we surveyed the protein abundance profiles linked to neurofilament bundle assembly, neuron projections, and neuronal cell body formation. These proteins exhibited increases in abundance level, yet we observed multiple patterns among the queried proteins. The data presented represent a rich resource for investigating temporal protein abundance changes in SH-SY5Y cells differentiated with retinoic acid. Moreover, the sample preparation and data acquisition strategies used here can be readily applied to any analogous cell line differentiation analysis.

Optimized Workflow for Multiplexed Phosphorylation Analysis of TMT-Labeled Peptides Using High-Field Asymmetric Waveform Ion Mobility Spectrometry.

Phosphorylation is a post-translational modification with a vital role in cellular signaling. Isobaric labeling-based strategies, such as tandem mass tags (TMT), can measure the relative phosphorylation states of peptides in a multiplexed format. However, the low stoichiometry of protein phosphorylation constrains the depth of phosphopeptide analysis by mass spectrometry. As such, robust and sensitive workflows are required. Here we evaluate and optimize high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) coupled to Orbitrap Tribrid mass spectrometers for the analysis of TMT-labeled phosphopeptides. We determined that using FAIMS-MS3 with three compensation voltages (CV) in a single method (e.g., CV = -40/-60/-80 V) maximizes phosphopeptide coverage while minimizing inter-CV overlap. Furthermore, consecutive analyses using MSA-CID (multistage activation collision-induced dissociation) and HCD (higher-energy collisional dissociation) fragmentation at the MS2 stage increases the depth of phosphorylation analysis. The methodology and results outlined herein provide a template for tailoring optimized FAIMS-based methods.

TKO6: A Peptide Standard To Assess Interference for Unit-Resolved Isobaric Labeling Platforms.

Protein abundance profiling using isobaric labeling is a well-established quantitative mass spectrometry technique. However, ratio distortion resulting from coisolated and cofragmented ions, commonly referred to as interference, remains a drawback of this strategy. Tribrid mass spectrometers, such as the Orbitrap Fusion and the Orbitrap Fusion Lumos with a triple mass analyzer configuration, facilitate methods (namely, SPS-MS3) that can help alleviate interference. However, few standards are available to measure interference and thereby aid in method development. Here we introduce the TKO6 standard that assesses ion interference and is designed specifically for data acquired at low (unit) mass resolution. We use TKO6 to compare interference in MS2- versus MS3-based quantitation methods, data acquisition methods of different lengths, and ion-trap-based tandem mass tag reporter ion analysis (IT-MS3) with conventional Orbitrap-based analysis (OT-MS3). We show that the TKO6 standard is a valuable tool for assessing quantification accuracy in isobaric-tag-based analyses.

Web-Based Search Tool for Visualizing Instrument Performance Using the Triple Knockout (TKO) Proteome Standard.

Multiplexing strategies are at the forefront of mass-spectrometry-based proteomics, with SPS-MS3 methods becoming increasingly commonplace. A known caveat of isobaric multiplexing is interference resulting from coisolated and cofragmented ions that do not originate from the selected precursor of interest. The triple knockout (TKO) standard was designed to benchmark data collection strategies to minimize interference. However, a limitation to its widespread use has been the lack of an automated analysis platform. We present a TKO Visualization Tool (TVT). The TVT viewer allows for automated, web-based, database searching of the TKO standard, returning traditional figures of merit, such as peptide and protein counts, scan-specific ion accumulation times, as well as the TKO-specific metric, the IFI (interference-free index). Moreover, the TVT viewer allows for plotting of two TKO standards to assess protocol optimizations, compare instruments, or measure degradation of instrument performance over time. We showcase the TVT viewer by probing the selection of (1) stationary phase resin, (2) MS2 isolation window width, and (3) number of synchronous precursor selection (SPS) ions for SPS-MS3 analysis. Using the TVT viewer will allow the proteomics community to search and compare TKO results to optimize user-specific data collection workflows.

Active Instrument Engagement Combined with a Real-Time Database Search for Improved Performance of Sample Multiplexing Workflows.

Quantitative proteomics employing isobaric reagents has been established as a powerful tool for biological discovery. Current workflows often utilize a dedicated quantitative spectrum to improve quantitative accuracy and precision. A consequence of this approach is a dramatic reduction in the spectral acquisition rate, which necessitates the use of additional instrument time to achieve comprehensive proteomic depth. This work assesses the performance and benefits of online and real-time spectral identification in quantitative multiplexed workflows. A Real-Time Search (RTS) algorithm was implemented to identify fragment spectra within milliseconds as they are acquired using a probabilistic score and to trigger quantitative spectra only upon confident peptide identification. The RTS-MS3 was benchmarked against standard workflows using a complex two-proteome model of interference and a targeted 10-plex comparison of kinase abundance profiles. Applying the RTS-MS3 method provided the comprehensive characterization of a 10-plex proteome in 50% less acquisition time. These data indicate that the RTS-MS3 approach provides dramatic performance improvements for quantitative multiplexed experiments.

TomahaqCompanion: A Tool for the Creation and Analysis of Isobaric Label Based Multiplexed Targeted Assays.

Triggered by Offset, Multiplexed, Accurate mass, High resolution, and Absolute Quantitation (TOMAHAQ) is a recently introduced targeted proteomics method that combines peptide and sample multiplexing. TOMAHAQ assays enable sensitive and accurate multiplexed quantification by implementing an intricate data collection scheme that comprises multiple MSn scans, mass inclusion lists, and data-driven filters. Consequently, manual creation of TOMAHAQ methods can be time-consuming and error prone, while the resulting TOMAHAQ data may not be compatible with common mass spectrometry analysis pipelines. To address these concerns we introduce TomahaqCompanion, an open-source desktop application that enables rapid creation of TOMAHAQ methods and analysis of TOMAHAQ data. Starting from a list of peptide sequences, a user can perform each step of TOMAHAQ assay development including (1) generation of priming run target list, (2) analysis of priming run data, (3) generation of TOMAHAQ method file, and (4) analysis and export of quantitative TOMAHAQ data. We demonstrate the flexibility of TomahaqCompanion by creating a variety of methods testing TOMAHAQ parameters (e.g., number of SPS notches, run length, etc.). Lastly, we analyze an interference sample comprising heavy yeast peptides, a standard human peptide mixture, TMT11-plex, and super heavy TMT (shTMT) isobaric labels to demonstrate ∼10-200 attomol limit of quantification within a complex background using TOMAHAQ.

Quantitative proteomic analysis of prostate tissue specimens identifies deregulated protein complexes in primary prostate cancer.

BACKGROUND: Prostate cancer (PCa) is the most frequently diagnosed non-skin cancer and a leading cause of mortality among males in developed countries. However, our understanding of the global changes of protein complexes within PCa tissue specimens remains very limited, although it has been well recognized that protein complexes carry out essentially all major processes in living organisms and that their deregulation drives the pathogenesis and progression of various diseases. METHODS: By coupling tandem mass tagging-synchronous precursor selection-mass spectrometry/mass spectrometry/mass spectrometry with differential expression and co-regulation analyses, the present study compared the differences between protein complexes in normal prostate, low-grade PCa, and high-grade PCa tissue specimens. RESULTS: Globally, a large downregulated putative protein-protein interaction (PPI) network was detected in both low-grade and high-grade PCa, yet a large upregulated putative PPI network was only detected in high-grade but not low-grade PCa, compared with normal controls. To identify specific protein complexes that are deregulated in PCa, quantified proteins were mapped to protein complexes in CORUM (v3.0), a high-quality collection of 4274 experimentally verified mammalian protein complexes. Differential expression and gene ontology (GO) enrichment analyses suggested that 13 integrin complexes involved in cell adhesion were significantly downregulated in both low- and high-grade PCa compared with normal prostate, and that four Prothymosin alpha (ProTα) complexes were significantly upregulated in high-grade PCa compared with normal prostate. Moreover, differential co-regulation and GO enrichment analyses indicated that the assembly levels of six protein complexes involved in RNA splicing were significantly increased in low-grade PCa, and those of four subcomplexes of mitochondrial complex I were significantly increased in high-grade PCa, compared with normal prostate. CONCLUSIONS: In summary, to the best of our knowledge, the study represents the first large-scale and quantitative, albeit indirect, comparison of individual protein complexes in human PCa tissue specimens. It may serve as a useful resource for better understanding the deregulation of protein complexes in primary PCa.

Multiplexed Isobaric Tag-Based Profiling of Seven Murine Tissues Following In Vivo Nicotine Treatment Using a Minimalistic Proteomics Strategy.

Nicotine is a major addictive compound in tobacco and a component of smoking-related products, such as e-cigarettes. Once internalized, nicotine can perturb many cellular pathways and can induce alterations in proteins across different cell types; however, the mechanisms thereof remain undetermined. The authors hypothesize that both tissue-specific and global protein abundance alterations result from nicotine exposure. Presented here is the first proteomic profiling of multiple tissues from mice treated orally with nicotine. Proteins extracted from seven tissues (brain, heart, kidney, liver, lung, pancreas, and spleen) from treated (n = 5) and untreated control (n = 5) mice are assembled into a TMT10-plex experiment. A minimalistic proteomics strategy is employed using TMT reagents efficiently and centrifugation-based reversed-phase columns to streamline sample preparation. Combined, over 11 000 non-redundant proteins from over 138 000 different peptides are quantified in seven TMT10-plex experiments. Between 7 and 126 proteins are significantly altered in tissues from nicotine-exposed mice, 11 which are altered in two or more tissues. Our data showcase the vast extent of nicotine exposure across murine tissue.

Isobaric Tag-Based Protein Profiling of a Nicotine-Treated Alpha7 Nicotinic Receptor-Null Human Haploid Cell Line.

Nicotinic acetylcholine receptors (nAChR), the primary cell surface targets of nicotine, have implications in various neurological disorders. Here we investigate the proteome-wide effects of nicotine on human haploid cell lines (wildtype HAP1 and α7KO-HAP1) to address differences in nicotine-induced protein abundance profiles between these cell lines. We performed an SPS-MS3-based TMT10-plex experiment arranged in a 2-3-2-3 design with two replicates of the untreated samples and three of the treated samples for each cell line. We quantified 8775 proteins across all ten samples, of which several hundred differed significantly in abundance. Comparing α7KO-HAP1 and HAP1wt cell lines to each other revealed significant protein abundance alterations; however, we also measured differences resulting from nicotine treatment in both cell lines. Among proteins with increased abundance levels due to nicotine treatment included those previously identified: APP, APLP2, and ITM2B. The magnitude of these changes was greater in HAP1wt compared to the α7KO-HAP1 cell line, implying a potential role for the α7 nAChR in HAP1 cells. Moreover, the data revealed that membrane proteins and proteins commonly associated with neurons were predominant among those with altered abundance. This study, which is the first TMT-based proteome profiling of HAP1 cells, defines further the effects of nicotine on non-neuronal cellular proteomes.

Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows.

High-throughput proteome profiling requires thorough optimization to achieve comprehensive analysis. We developed a filter aided sample preparation (FASP)-like, detergent-free method, termed Filter-Based Protein Digestion (FPD). We compared FPD to protein extraction methods commonly used in isobaric tag-based proteome profiling, namely trichloroacetic acid (TCA) and chloroform-methanol (C-M) precipitation. We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma cell line for parallel protein processing with TCA (n = 3), C-M (n = 2), and FPD using either 10 kDa (n = 3) or 30 kDa (n = 3) molecular weight cutoff membranes. We labeled each sample with tandem mass tag (TMT) reagents to construct a TMT11-plex experiment. In total, 8654 proteins were quantified across all samples. Pairwise comparisons showed very little deviation for individual protein abundance measurements between the two FPD methods, whereas TCA and FPD showed the most difference. Specifically, membrane proteins were more readily quantified when samples were processed using TCA precipitation than other methods tested. However, globally, only 4% of proteins differed greater than 4-fold in the most divergent pair of protein extraction methods (i.e., FPD10 and TCA). We conclude that the detergent-free FPD strategy, particularly using the faster-flowing 30 kDa filter, is a seamless alteration to high-throughput TMT workflows.

Parsing and Quantification of Raw Orbitrap Mass Spectrometer Data Using RawQuant.

Effective analysis of protein samples by mass spectrometry (MS) requires careful selection and optimization of a range of experimental parameters. As the output from the primary detection device, the "raw" MS data file can be used to gauge the success of a given sample analysis. However, the closed-source nature of the standard raw MS file can complicate effective parsing of the data contained within. To ease and increase the range of analyses possible, the RawQuant tool was developed to enable parsing of raw MS files derived from Thermo Orbitrap instruments to yield meta and scan data in an openly readable text format. RawQuant can be commanded to export user-friendly files containing MS1, MS2, and MS3 metadata as well as matrices of quantification values based on isobaric tagging approaches. In this study, the utility of RawQuant is demonstrated in several scenarios: (1) reanalysis of shotgun proteomics data for the identification of the human proteome, (2) reanalysis of experiments utilizing isobaric tagging for whole-proteome quantification, and (3) analysis of a novel bacterial proteome and synthetic peptide mixture for assessing quantification accuracy when using isobaric tags. Together, these analyses successfully demonstrate RawQuant for the efficient parsing and quantification of data from raw Thermo Orbitrap MS files acquired in a range of common proteomics experiments. In addition, the individual analyses using RawQuant highlights parametric considerations in the different experimental sets and suggests targetable areas to improve depth of coverage in identification-focused studies and quantification accuracy when using isobaric tags.

Nicotine-induced protein expression profiling reveals mutually altered proteins across four human cell lines.

Mass spectrometry-based proteomic strategies can profile the expression level of proteins in response to external stimuli. Nicotine affects diverse cellular pathways, however, the nicotine-induced alterations on the global proteome across human cell lines have not been fully elucidated. We measured perturbations in protein levels resulting from nicotine treatment in four cell lines-HEK, HeLa, PaSC, and SH-SY5Y-in a single experiment using tandem mass tags (TMT10-plex) and high-resolution mass spectrometry. We quantified 8590 proteins across all cell lines. Of these, nicotine increased the abundance of 31 proteins 1.5-fold or greater in all cell lines. Likewise, considering proteins with altered levels in at least three of the four cell lines, 64 were up-regulated, while one was down-regulated. Gene ontology analysis revealed that ∼40% of these proteins were membrane bound, and functioned in transmembrane signaling and receptor activity. We highlighted proteins, including APP, APLP2, LAPTM4B, and NCOA4, which were dysregulated by nicotine in all cell lines investigated and may have implications in downstream signaling pathways, particularly autophagy. Using the outlined methodology, studies in additional (including primary) cell lines will provide further evidence that alterations in the levels of these proteins are indeed a general response to nicotine and thereby merit further investigation.

Multiplexed Phosphoproteomic Profiling Using Titanium Dioxide and Immunoaffinity Enrichments Reveals Complementary Phosphorylation Events.

A comprehensive view of protein phosphorylation remains an unmet challenge in the field of cell biology. Mass spectrometry-based proteomics is one of the most promising approaches for identifying thousands of phosphorylation events in a single experiment, yet the full breadth of the phosphoproteome has yet to be elucidated. In this article, we examined the complementarity of two methods for phosphopeptide enrichment based on either titanium dioxide (TiO2) enrichment or phosphorylation motif-specific immunoaffinity precipitation (IAP) with four different antibodies. Each method identified nearly 2000 phosphoproteins. However, distinct populations of phosphopeptides were observed. Despite quantifying over 10 000 unique phosphorylation events using TiO2 and over 3900 with IAP, less than 5% of the sites were in common. Agreeing with published literature, the ratio of pS:pT:pY phosphorylation for the TiO2-enriched data set approximated 90:10:<1. In contrast, that ratio for the combined IAP data sets was 51:29:20. These differences not only suggest the complementarity between multiple enrichment methods but also emphasize their collective importance in obtaining a comprehensive view of the phosphoproteome.

Improved Method for Determining Absolute Phosphorylation Stoichiometry Using Bayesian Statistics and Isobaric Labeling.

Phosphorylation stoichiometry, or occupancy, is one element of phosphoproteomics that can add useful biological context (Gerber et al. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 6940-5). We previously developed a method to assess phosphorylation stoichiometry on a proteome-wide scale (Wu et al. Nat. Methods 2011, 8, 677-83). The stoichiometry calculation relies on identifying and measuring the levels of each nonphosphorylated counterpart peptide with and without phosphatase treatment. The method, however, is problematic in that low stoichiometry phosphopeptides can return negative stoichiometry values if measurement error is larger than the percent stoichiometry. Here, we have improved the stoichiometry method through the use of isobaric labeling with 10-plex TMT reagents. In this way, five phosphatase treated and five untreated samples are compared simultaneously so that each stoichiometry is represented by five ratio measurements with no missing values. We applied the method to determine basal stoichiometries of HCT116 cells growing in culture. With this method, we analyzed five biological replicates simultaneously with no need for phosphopeptide enrichment. Additionally, we developed a Bayesian model to estimate phosphorylation stoichiometry as a parameter confined to an interval between 0 and 1 implemented as an R/Stan script. Consequently, both point and interval estimates are consistent with the plausible range of values for stoichiometry. Finally, we report absolute stoichiometry measurements with credible intervals for 6772 phosphopeptides containing at least a single phosphorylation site.