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.

Overcoming the detrimental O-acylation in TMTpro labeling improves the proteome depth and quantification precision.

BACKGROUND: With the advent of proline-based reporter isobaric Tandem Mass Tag (TMTpro) reagents, the sample multiplexing capacity of tandem mass tags (TMTs) has been expanded, and up to 18 samples can be quantified in a multiplexed manner. Like classic TMT reagents, TMTpro reagents contain a tertiary amine group, which markedly enhances their reactivity toward hydroxyl groups and results in O-acylation of serine, threonine and tyrosine residues. This overlabeling significantly compromises proteome analysis in terms of depth and precision. In particular, the reactivity of hydroxyl-containing residues can be dramatically enhanced when coexisting with a histidine in the same peptides, leading to a severe systematic bias against the analysis of these peptides. Although some protocols using a reduced molar excess of TMT under alkaline conditions can alleviate overlabeling of histidine-free peptides to some extent, they have a limited effect on histidyl- and hydroxyl-containing peptides. RESULTS: Here, we report a novel TMTpro labeling method that overcomes detrimental overlabeling while providing high labeling efficiency of amines. Additionally, our method is cost-effective, as it requires only half the amount of TMTpro reagents recommended by the reagent manufacturer. In a deep-scale analysis of a yeast/human two-proteome model sample, we compared our method with a typical alkaline labeling method using a reduced molar excess of TMTpro. Even at a depth of over 10,000 proteins, our method detected 23.7% more unique peptides and 8.7% more protein groups compared to the alkaline labeling method. Moreover, our method significantly improved the quantitative precision due to the reduced variability in labeling and increased protein sequence coverage. This substantially enhanced the statistical power of our method for detecting differentially abundant proteins, providing an average of 13% more yeast proteins that reached statistical significance. SIGNIFCANCE: We presented a novel TMTpro labeling method that overcomes the detrimental O-acylation and thus significantly improves the depth and quantitative precision for proteome analysis.

Spectral library search for improved TMTpro labelled peptide assignment in human plasma proteomics.

Clinical biomarker discovery is often based on the analysis of human plasma samples. However, the high dynamic range and complexity of plasma pose significant challenges to mass spectrometry-based proteomics. Current methods for improving protein identifications require laborious pre-analytical sample preparation. In this study, we developed and evaluated a TMTpro-specific spectral library for improved protein identification in human plasma proteomics. The library was constructed by LC-MS/MS analysis of highly fractionated TMTpro-tagged human plasma, human cell lysates, and relevant arterial tissues. The library was curated using several quality filters to ensure reliable peptide identifications. Our results show that spectral library searching using the TMTpro spectral library improves the identification of proteins in plasma samples compared to conventional sequence database searching. Protein identifications made by the spectral library search engine demonstrated a high degree of complementarity with the sequence database search engine, indicating the feasibility of increasing the number of protein identifications without additional pre-analytical sample preparation. The TMTpro-specific spectral library provides a resource for future plasma proteomics research and optimization of search algorithms for greater accuracy and speed in protein identifications in human plasma proteomics, and is made publicly available to the research community via ProteomeXchange with identifier PXD042546.

Adapting an Isobaric Tag-Labeled Yeast Peptide Standard to Develop Targeted Proteomics Assays.

Targeted proteomics strategies present a streamlined hypothesis-driven approach to analyze specific sets of pathways or disease related proteins. goDig is a quantitative, targeted tandem mass tag (TMT)-based assay that can measure the relative abundance differences for hundreds of proteins directly from unfractionated mixtures. Specific protein groups or entire pathways of up to 200 proteins can be selected for quantitative profiling, while leveraging sample multiplexing permits the simultaneous analysis of up to 18 samples. Despite these benefits, implementing goDig is not without challenges, as it requires access to an instrument application programming interface (iAPI), an elution order and spectral library, a web-based method builder, and dedicated companion software. In addition, the absence of an example test assay may dissuade researchers from testing or implementing goDig. Here, we repurpose the TKO11 standard─which is commercially available but may also be assembled in-lab─and establish it as a de facto test assay for goDig. We build a proteome-wide goDig yeast library, quantify protein expression across several gene ontology (GO) categories, and compare these results to a fully fractionated yeast gold-standard data set. Essentially, we provide a guide detailing the goDig-based quantification of TKO11, which can also be used as a template for user-defined assays in other species.

Proteome-Wide Profiling Using Sample Multiplexing of a Human Cell Line Treated with Cannabidiol (CBD) and Tetrahydrocannabinol (THC).

Cannabis has been used historically for both medicinal and recreational purposes, with the most notable cannabinoids being cannabidiol (CBD) and tetrahydrocannabinol (THC). Although their therapeutic effects have been well studied and their recreational use is highly debated, the underlying mechanisms of their biological effects remain poorly defined. In this study, we use isobaric tag-based sample multiplexed proteome profiling to investigate protein abundance differences in the human neuroblastoma SH-SY5Y cell line treated with CBD and THC. We identified significantly regulated proteins by each treatment and performed a pathway classification and associated protein-protein interaction analysis. Our findings suggest that these treatments may lead to mitochondrial dysfunction and induce endoplasmic reticulum stress. These data can potentially be interrogated further to investigate the potential role of CBD and THC in various biological and disease contexts, providing a foundation for future studies.

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.

Comparison of the Proteomes and Phosphoproteomes of S. cerevisiae Cells Harvested with Different Strategies.

The budding yeast Saccharomyces cerevisiae is a powerful model system that is widely used to investigate many cellular processes. The harvesting of yeast cells is the first step in almost every experimental procedure. Here, yeast cells are isolated from their growth medium, collected, and used for successive experiments or analysis. The two most common methods to harvest S. cerevisiae are centrifugation and filtration. Understanding if and how centrifugation and filtration affect yeast physiology is essential with respect to downstream data interpretation. Here, we profile and compare the proteomes and the phosphoproteomes, using isobaric label-based quantitative mass spectrometry, of three common methods used to harvest S. cerevisiae cells: low-speed centrifugation, high-speed centrifugation, and filtration. Our data suggest that, while the proteome was stable across the tested conditions, hundreds of phosphorylation events were different between centrifugation and filtration. Our analysis shows that, under our experimental conditions, filtration may cause both cell wall and osmotic stress at higher levels compared to centrifugation, implying harvesting-method-specific stresses. Thus, considering that the basal activation levels of specific stresses may differ under certain harvesting conditions is an important, but often overlooked, aspect of experimental design.

Comparative Proteomic Analysis of Two Commonly Used Laboratory Yeast Strains: W303 and BY4742.

The yeast Saccharomyces cerevisiae is a powerful model system that is often used to expand our understanding of cellular processes and biological functions. Although many genetically well-characterized laboratory strains of S. cerevisiae are available, they may have different genetic backgrounds which can confound data interpretation. Here, we report a comparative whole-proteome analysis of two common laboratory yeast background strains, W303 and BY4742, in both exponential and stationary growth phases using isobaric-tag-based mass spectrometry to highlight differences in proteome complexity. We quantified over 4400 proteins, hundreds of which showed differences in abundance between strains and/or growth phases. Moreover, we used proteome-wide protein abundance to profile the mating type of the strains used in the experiment, the auxotrophic markers, and associated metabolic pathways, as well as to investigate differences in particular classes of proteins, such as the pleiotropic drug resistance (PDR) proteins. This study is a valuable resource that offers insight into mechanistic differences between two common yeast background strains and can be used as a guide to select a background that is best suited for addressing a particular biological question.

Proteome-wide abundance profiling of yeast deletion strains for GET pathway members using sample multiplexing.

The GET pathway is associated with post-translational delivery of tail-anchored (TA) proteins to the endoplasmic reticulum (ER) in yeast, as well as other eukaryotes. Moreover, dysfunction of the GET pathway has been associated with various pathological conditions (i.e., neurodegenerative disorders, cardiovascular ailments, and protein misfolding diseases). In this study, we used yeast deletion strains of Get complex members (specifically, Get1, Get2, Get3, Get4, and Get5) coupled with sample multiplexing-based quantitative mass spectrometry to profile protein abundance on a proteome-wide scale across the five individual deletion strains. Our dataset consists of over 4500 proteins, which corresponds to >75% of the yeast proteome. The data reveal several dozen proteins that are differentially abundant in one or more deletion strains, some of which are membrane-associated, yet the abundance of many TA proteins remained unchanged. This study provides valuable insights into the roles of these Get genes, and the potential for alternative pathways which help maintain cellular function despite the disruption of the GET pathway.

Enriching Cysteine-Containing Peptides Using a Sulfhydryl-Reactive Alkylating Reagent with a Phosphonic Acid Group and Immobilized Metal Affinity Chromatography.

The reduction of disulfide bonds and their subsequent alkylation are commonplace in typical proteomics workflows. Here, we highlight a sulfhydryl-reactive alkylating reagent with a phosphonic acid group (iodoacetamido-LC-phosphonic acid, 6C-CysPAT) that facilitates the enrichment of cysteine-containing peptides for isobaric tag-based proteome abundance profiling. Specifically, we profile the proteome of the SH-SY5Y human cell line following 24 h treatments with two proteasome inhibitors (bortezomib and MG-132) in a tandem mass tag (TMT)pro9-plex experiment. We acquire three datasets─(1) Cys-peptide enriched, (2) the unbound complement, and (3) the non-depleted control─and compare the peptides and proteins quantified in each dataset, with emphasis on Cys-containing peptides. The data show that enrichment using 6C-Cys phosphonate adaptable tag (6C-CysPAT) can quantify over 38,000 Cys-containing peptides in 5 h with >90% specificity. In addition, our combined dataset provides the research community with a resource of over 9900 protein abundance profiles exhibiting the effects of two different proteasome inhibitors. Overall, the seamless incorporation of alkylation by 6C-CysPAT into a current TMT-based workflow permits the enrichment of a Cys-containing peptide subproteome. The acquisition of this "mini-Cys" dataset can be used to preview and assess the quality of a deep, fractionated dataset.

Spin column-based peptide fractionation alternatives for streamlined tandem mass tag (SL-TMT) sample processing.

Fractionation is essential to achieving deep proteome coverage for sample multiplexing experiments where currently up to 18 samples can be analyzed concurrently. However, peptide fractionation (i.e., upstream of LC-MS/MS analysis) with a liquid chromatography system constrains sample processing as only a single sample can be fractionated at once. Here, we highlight the use of spin column-based methods which permit multiple multiplexed samples to be fractionated simultaneously. These methods require only a centrifuge and eliminate the need for a dedicated liquid chromatography system. We investigate peptide fractionation with strong anion exchange (SAX) and high-pH reversed phase (HPRP) spin columns, as well as a combination of both. In two separate experiments, we acquired deep proteome coverage (>8000 quantified proteins), while starting with <25 µg of protein per channel. Our datasets showcase the proteome alterations in two human cell lines resulting from treatment with inhibitors acting on the ubiquitin-proteasome system. We recommend this spin column-based peptide fractionation strategy for high-throughput screening applications or whenever a liquid chromatograph is not readily available. SIGNIFICANCE: Fractionation is a means to achieve deep proteome coverage for global proteomics analysis. Typical liquid chromatography systems may be a prohibitive expense for many laboratories. Here, we investigate prefractionation with strong anion exchange (SAX) and high-pH reversed phase (HPRP) spin columns, as well as a combination of both, as peptide fractionation methods. These spin columns have advantages over liquid chromatography systems, which include relative affordability, higher throughput capability, no carry over, and fewer potential instrument-related malfunctions. In two separate experiments, we acquired deep proteome coverage (>8000 quantified proteins), thereby showing the utility of each or a combination of both spin columns for global proteome analysis.

High-Throughput and In-Depth Proteomic Profiling of 5 µL Plasma and Serum Using TMTpro 16-Plex.

High-throughput and in-depth proteomic analysis of plasma and serum samples remains challenging due to the presence of multiple high-abundance proteins. Here, we provide a detailed protocol for proteomic analysis of serum and plasma specimens using a high-abundance protein depletion kit and TMTpro 16-plex reagents. This method requires only 5 µL serum or plasma, identifying and quantifying about 1000 proteins. A batch of 16 samples can be processed in 36 h. On average, each sample consumes about 1.5 h of mass spectrometer instrument time. Overall, our method can identify proteins across six orders of magnitude with high reproducibility (CV < 20%) using a shorter instrument time and less sample volume compared to existing methods. Thus, the method is suitable to be applied to large-scale proteomic studies.

SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics.

Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.

Quantitative proteome remodeling characterization of two human reference pluripotent stem cell lines during neurogenesis and cardiomyogenesis.

Human pluripotent stem cells (PSCs) have become popular tools within the research community to study developmental and model diseases. While many induced-PSCs (iPSCs) from various genetic background sources are currently available, scientific advancement has been hampered by the considerable phenotypic variations observed between different iPSC lines. A recent collaborative effort selected a novel iPSC line to address this and encourage the adoption of a standardized iPSC line termed KOLF2.1J. Here, leveraging the multiplexing power of isobaric labeling, we systematically investigate, at the 10k proteome level, the relative protein abundance profiles of the KOLF2.1J reference iPSC line upon two distinct cell state differentiation trajectories. In addition, we side-by-side systematically compare this line with the H9 line, an established embryonically derived PSC line that we previously characterized. We noticed differences in the basal proteome of the two cell lines and highlighted the differentially expressed proteins. While the difference between the cell line's proteome subsisted upon differentiation, the global proteome remodeling trajectory was highly similar during the tested differentiation routes. We thus conclude that the KOLF2.1J line performs well at the proteome level upon the neuro and cardiomyogenesis differentiation protocol used. We believe this dataset will serve as a resource of value for the research community.

Predicting fragment intensities and retention time of iTRAQ- and TMTPro-labeled peptides with Prosit-TMT.

Isobaric labeling increases the throughput of proteomics by enabling the parallel identification and quantification of peptides and proteins. Over the past decades, a variety of isobaric tags have been developed allowing the multiplexed analysis of up to 18 samples. However, experiments utilizing such tags often exhibit reduced identification rates and thus show decreased analytical depth. Re-scoring has been shown to rescue otherwise missed identifications but was not yet systematically applied on isobarically labeled data. Because iTRAQ 4/8-plex and the recently released TMTpro 16/18-plex share similar characteristics with TMT 6/10/11-plex, we hypothesized that Prosit-TMT, trained exclusively on 6/10/11-plex labeled peptides, may be applicable to these isobaric labeling strategies as well. To investigate this, we re-analyzed nine publicly available datasets covering iTRAQ and TMTpro labeling for samples with human and mouse origin. We highlight that Prosit-TMT shows remarkably good performance when comparing experimentally acquired and predicted fragmentation spectra (R of 0.84 - 0.9) and retention times (ΔRT95% of 3% - 10% gradient time) of peptides. Furthermore, re-scoring substantially increases the number of confidently identified spectra, peptides, and proteins.

Profiling Yeast Deletion Strains Using Sample Multiplexing and Network-Based Analyses.

The yeast, Saccharomyces cerevisiae, is a widely used model system for investigating conserved biological functions and pathways. Advancements in sample multiplexing have facilitated the study of the yeast proteome, yet many yeast proteins remain uncharacterized or only partially characterized. Yeast deletion strain collections are powerful resources for yeast proteome studies, uncovering the effects of gene function, genetic interactions, and cellular stresses. As complex biological systems cannot be understood by simply analyzing the individual components, a systems approach is often required in which a protein is represented as a component of large, interacting networks. Here, we evaluate the current state of yeast proteome analysis using isobaric tag-based sample multiplexing (TMTpro16) to profile the proteomes of 75 yeast deletion strains for which we measured the abundance of nearly 5000 proteins. Using statistical approaches, we highlighted covariance and regulation subnetworks and the enrichment of gene ontology classifications for covarying and coregulated proteins. This dataset presents a resource that is amenable to further data mining to study individual deletion strains, pathways, proteins, and/or interactions thereof while serving as a template for future network-based investigations using yeast deletion strain collections.

Retention Time Prediction for TMT-Labeled Peptides in Proteomic LC-MS Experiments.

We present the first detailed study of chromatographic behavior of peptides labeled with tandem mass tags (TMT and TMTpro) in 2D LC for proteomic applications. Carefully designed experimental procedures have permitted generating data sets of over 100,000 nonlabeled and TMT-labeled peptide pairs for the low pH RP in the second separation dimension and data sets of over 10,000 peptide pairs for high-pH RP, HILIC (amide and silica), and SCX separations in the first separation dimension. The average increase in peptide RPLC (0.1% formic acid) retention upon TMT labeling was found to be 3.3% acetonitrile (linear water/acetonitrile gradients), spanning a range of -4 to 10.3%. In addition to the bulk peptide properties such as length, hydrophobicity, and the number of labeled residues, we found several sequence-dependent features mostly associated with differences in N-terminal chemistry. The behavior of TMTpro-labeled peptides was found to be very similar except for a slightly higher hydrophobicity: an average retention shift of 3.7% acetonitrile. The respective versions of the sequence-specific retention calculator (SSRCalc) model have been developed to accommodate both TMT chemistries, showing identical prediction accuracy (R2 ∼ 0.98) for labeled and nonlabeled peptides. Higher retention for TMT-labeled peptides was observed for high-pH RP and HILIC separations, while SCX selectivity remained virtually unchanged.

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.

Proteome-wide mapping of short-lived proteins in human cells.

Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.

Strategies for mass spectrometry-based phosphoproteomics using isobaric tagging.

INTRODUCTION: Protein phosphorylation is a primary mechanism of signal transduction in cellular systems. Isobaric tagging can be used to investigate alterations in phosphorylation events in sample multiplexing experiments where quantification extends across all conditions. As such, innovations in tandem mass tag methods can facilitate the expansion of the depth and breadth of phosphoproteomic analyses. AREAS COVERED: This review discusses the current state of tandem mass tag-centric phosphoproteomics and highlights advances in reagent chemistry, instrumentation, data acquisition, and data analysis. We stress that approaches for phosphoproteomic investigations require high-specificity enrichment, sensitive detection, and accurate phosphorylation site localization. EXPERT OPINION: Tandem mass tag-centric phosphoproteomics will continue to be an important conduit for our understanding of signal transduction in living organisms. We anticipate that progress in phosphopeptide enrichment methodologies, enhancements in instrumentation and data acquisition technologies, and further refinements in analytical strategies will be key to the discovery of biologically relevant findings from phosphoproteomics studies.