Sample Multiplexing Research Articles

TMTpro-18plex: The Expanded and Complete Set of TMTpro Reagents for Sample Multiplexing

The development of the TMTpro-16plex series expanded the breadth of commercial isobaric tagging reagents by nearly 50% over classic TMT-11plex. In addition to the described 16plex reagents, the proline-based TMTpro molecule can accommodate two additional combinations of heavy carbon and nitrogen isotopes. Here, we introduce the final two labeling reagents, TMTpro-134C and TMTpro-135N, which permit the simultaneous global protein profiling of 18 samples with essentially no missing values. For example, six conditions with three biological replicates can now be perfectly accommodated. We showcase the 18plex reagent set by profiling the proteome and phosphoproteome of a pair of isogenic mammary epithelial cell lines under three conditions in triplicate. We compare the depth and quantitative performance of this data set with a TMTpro-16plex experiment in which two samples were omitted. Our analysis revealed similar numbers of quantified peptides and proteins, with high quantitative correlation. We interrogated further the TMTpro-18plex data set by highlighting changes in protein abundance profiles under different conditions in the isogenic cell lines. We conclude that TMTpro-18plex further expands the sample multiplexing landscape, allowing for complex and innovative experimental designs.

Temporal proteomic changes induced by nicotine in human cells: A quantitative proteomics approach

Nicotine is a prominent active compound in tobacco and many smoking cessation products. Some of the biological effects of nicotine are well documented in in vitro and in vivo systems; however, data are scarce concerning the time-dependent changes on protein and phosphorylation events in response to nicotine. Here, we profiled the proteomes of SH-SY5Y and A549 cell lines subjected to acute (15 min, 1 h and 4 h) or chronic (24 h, 48 h) nicotine exposures. We used sample multiplexing (TMTpro16) and quantified more than 9000 proteins and over 7000 phosphorylation events per cell line. Among our findings, we determined a decrease in mitochondrial protein abundance for SH-SY5Y, while we detected alterations in several immune pathways, such as the complement system, for A549 following nicotine treatment. We also explored the proposed association between smoking (specifically nicotine) and SARS-CoV2. Here, we found several host proteins known to interact with viral proteins that were affected by nicotine in a time dependent manner. This dataset can be mined further to investigate the potential role of nicotine in different biological contexts. SignificanceSmoking is a major public health issue that is associated with several serious chronic, yet preventable diseases, including stroke, heart disease, type 2 diabetes, cancer, and susceptibility to infection. Tobacco smoke is a complex mixture of thousands of different compounds, among which nicotine is the main addictive compound. The biological effects of nicotine have been reported in several models, however very little data are available concerning the temporal proteomic and phosphoproteomic changes in response to nicotine. Here, we provide a dataset exploring the potential role of nicotine on different biological processes over time, including implications in the study of SARS-CoV2.

A global live cell barcoding approach for multiplexed mass cytometry profiling of mouse tumors.

With the advent of cancer immunology, mass cytometry has been increasingly employed to characterize the responses to cancer therapies and the tumor microenvironment (TME). One of its most notable applications is efficient multiplexing of samples into batches by dedicating a number of metal isotope channels to barcodes, enabling robust data acquisition and analysis. Barcoding is most effective when markers are present in all cells of interest. While CD45 has been shown to be a reliable marker for barcoding all immune cells in a given sample, a strategy to reliably barcode mouse cancer cells has not been demonstrated. To this end, we identified CD29 and CD98 as markers widely expressed by commonly used mouse cancer cell lines. We conjugated anti-CD29 and anti-CD98 antibodies to cadmium or indium metals and validated their utility in 10-plex barcoding of live cells. Finally, we established a potentially novel barcoding system incorporating the combination of CD29, CD98, and CD45 to multiplex 10 tumors from s.c. MC38 and KPC tumor models, while successfully recapitulating the known contrast in the PD1-PDL1 axis between the 2 models. The ability to barcode tumor cells along with immune cells empowers the interrogation of the tumor-immune interactions in mouse TME studies.

CASB: a concanavalin A‐based sample barcoding strategy for single‐cell sequencing

Sample multiplexing facilitates single‐cell sequencing by reducing costs, revealing subtle difference between similar samples, and identifying artifacts such as cell doublets. However, universal and cost‐effective strategies are rather limited. Here, we reported a concanavalin A‐based sample barcoding strategy (CASB), which could be followed by both single‐cell mRNA and ATAC (assay for transposase‐accessible chromatin) sequencing techniques. The method involves minimal sample processing, thereby preserving intact transcriptomic or epigenomic patterns. We demonstrated its high labeling efficiency, high accuracy in assigning cells/nuclei to samples regardless of cell type and genetic background, and high sensitivity in detecting doublets by three applications: 1) CASB followed by scRNA‐seq to track the transcriptomic dynamics of a cancer cell line perturbed by multiple drugs, which revealed compound‐specific heterogeneous response; 2) CASB together with both snATAC‐seq and scRNA‐seq to illustrate the IFN‐γ‐mediated dynamic changes on epigenome and transcriptome profile, which identified the transcription factor underlying heterogeneous IFN‐γ response; and 3) combinatorial indexing by CASB, which demonstrated its high scalability.

Advances in quantitative high-throughput phosphoproteomics with sample multiplexing.

Eukaryotic protein phosphorylation modulates nearly every major biological process. Phosphorylation regulates protein activity, mediates cellular signal transduction, and manipulates cellular structure. Consequently, the dysregulation of kinase and phosphatase pathways has been linked to a multitude of diseases. Mass spectrometry-based proteomic techniques are increasingly used for the global interrogation of perturbations in phosphorylation-based cellular signaling. Strategies for studying phosphoproteomes require high-specificity enrichment, sensitive detection, and accurate localization of phosphorylation sites with advanced LC-MS/MS techniques and downstream informatics. Sample multiplexing with isobaric tags has also been integral to recent advancements in throughput and sensitivity for phosphoproteomic studies. Each of these facets of phosphoproteomics analysis present distinct challenges and thus opportunities for improvement and innovation. Here, we review current methodologies, explore persistent challenges, and discuss the outlook for isobaric tag-based quantitative phosphoproteomic analysis.

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.

No detectable alloreactive transcriptional responses under standard sample preparation conditions during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells

BackgroundSingle-cell RNA sequencing (scRNA-seq) provides high-dimensional measurements of transcript counts in individual cells. However, high assay costs and artifacts associated with analyzing samples across multiple sequencing runs limit the study of large numbers of samples. Sample multiplexing technologies such as MULTI-seq and antibody hashing using single-cell multiplexing kit (SCMK) reagents (BD Biosciences) use sample-specific sequence tags to enable individual samples to be sequenced in a pooled format, markedly lowering per-sample processing and sequencing costs while minimizing technical artifacts. Critically, however, pooling samples could introduce new artifacts, partially negating the benefits of sample multiplexing. In particular, no study to date has evaluated whether pooling peripheral blood mononuclear cells (PBMCs) from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures.ResultsHere, we applied the 10x Genomics scRNA-seq platform to MULTI-seq and/or SCMK-labeled PBMCs from a single donor with and without pooling with PBMCs from unrelated donors for 30 min at 4 °C. We did not detect any alloreactivity signal between mixed and unmixed PBMCs across a variety of metrics, including alloreactivity marker gene expression in CD4+ T cells, cell type proportion shifts, and global gene expression profile comparisons using Gene Set Enrichment Analysis and Jensen-Shannon Divergence. These results were additionally mirrored in publicly-available scRNA-seq data generated using a similar experimental design. Moreover, we identified confounding gene expression signatures linked to PBMC preparation method (e.g., Trima apheresis), as well as SCMK sample classification biases against activated CD4+ T cells which were recapitulated in two other SCMK-incorporating scRNA-seq datasets.ConclusionsWe demonstrate that (i) mixing PBMCs from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) does not cause an allogeneic response, and (ii) that Trima apheresis and PBMC sample multiplexing using SCMK reagents can introduce undesirable technical artifacts into scRNA-seq data. Collectively, these observations establish important benchmarks for future cross-sectional immunological scRNA-seq experiments.

Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries.

Current methods used for measuring amino acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts and software to boost data acquisition in real time on the mass spectrometer. Our method, streamlined cysteine activity-based protein profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites at 18 min per compound. We applied it to identify proteome-wide targets of covalent inhibitors to mutant Kirsten rat sarcoma (KRAS)G12C and Bruton's tyrosine kinase (BTK). In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines, which includes >20,000 cysteines from >6,000 proteins per line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.

Sensitive and Quantitative Detection of MHC-I Displayed Neoepitopes Using a Semiautomated Workflow and TOMAHAQ Mass Spectrometry

Advances in several key technologies, including MHC peptidomics, have helped fuel our understanding of basic immune regulatory mechanisms and the identification of T cell receptor targets for the development of immunotherapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables characterization of dynamic changes in the ligandome due to cellular perturbations. However, the current multistep analytical process is challenging, and improvements in throughput and reproducibility would enable rapid characterization of multiple conditions in parallel. Here, we describe a robust and quantitative method whereby peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines such as MC38, can be enriched in a semiautomated fashion on reusable, dry-storage, customized antibody cartridges. Using this method, a researcher, with very little hands-on time and in a single day, can perform up to 96 simultaneous enrichments at a similar level of quality as a manual workflow. TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification), a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity, was employed to characterize neoepitopes displayed on MHC-I by tumor cells and to quantitatively assess the influence of neoantigen expression and induced degradation on neoepitope presentation. This unique combination of robust semiautomated MHC-I peptide isolation and high-throughput multiplexed targeted quantitation allows for both the routine analysis of >4000 unique MHC-I peptides from 250 million cells using nontargeted methods, as well as quantitative sensitivity down to the low amol/μl level using TOMAHAQ targeted MS.

Automating UbiFast for High-throughput and Multiplexed Ubiquitin Enrichment

Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites are necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-ϵ-GG peptides are enriched with anti-K-ε-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-ε-GG antibody (mK-ε-GG) and a magnetic particle processor. We report the identification of ∼20,000 ubiquitylation sites from a TMT10-plex with 500 μg input per sample processed in ∼2 h. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility, and significantly reduced processing time. The automated method also significantly reduced variability across process replicates compared with the manual method. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of the method to profile small amounts of tissue using breast cancer patient–derived xenograft (PDX) tissue samples.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT).

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

Phosphoproteomic quantitation and causal analysis reveal pathways in GPVI/ITAM-mediated platelet activation programs

AbstractPlatelets engage cues of pending vascular injury through coordinated adhesion, secretion, and aggregation responses. These rapid, progressive changes in platelet form and function are orchestrated downstream of specific receptors on the platelet surface and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) detected >3000 significant (false discovery rate < 0.05) phosphorylation events on >1300 proteins over conditions initiating and progressing GPVI-mediated platelet activation. With literature-guided causal inference tools, >300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (ie, FcRγ, Syk, PLCγ2, PKCδ, DAPP1). Through signaling validation studies and functional screening, other less-characterized targets were also considered within the context of GPVI/ITAM pathways, including Ras/MAPK axis proteins (ie, KSR1, SOS1, STAT1, Hsp27). Highly regulated GPVI/ITAM targets out of context of curated knowledge were also illuminated, including a system of >40 Rab GTPases and associated regulatory proteins, where GPVI-mediated Rab7 S72 phosphorylation and endolysosomal maturation were blocked by TAK1 inhibition. In addition to serving as a model for generating and testing hypotheses from omics datasets, this study puts forth a means to identify hemostatic effectors, biomarkers, and therapeutic targets relevant to thrombosis, vascular inflammation, and other platelet-associated disease states.

Derivatization-based sample-multiplexing for enhancing throughput in liquid chromatography/tandem mass spectrometry quantification of metabolites: an overview

The quantification of metabolites in various samples, including body fluids, tissues, cells, and foodstuffs, contributes to our understanding of their biological activities and roles in the body, diagnosis for many diseases, drug and biomarker discovery, and many aspects of human health. Liquid chromatography (LC)/tandem mass spectrometry (MS/MS) is the most powerful and reliable methodology for the quantification of metabolites due to its high specificity and sensitivity, and broad coverage of various compounds. Derivatization often makes the quantification power of LC/MS/MS stronger due to the desirable LC behavior and enhanced MS/MS detectability of the derivatized metabolites. On the other hand, LC/MS/MS-based quantification has room for improvement regarding its analysis throughput. Derivatization is also a promising approach to overcome this drawback; the multiplexing of samples in the same LC/MS/MS injection, which is achieved by derivatization of multiple samples with multiple well-designed reagents, can enhance the throughput. Based on this background, this article reviews the derivatization-based sample-multiplexing strategy, especially the characteristics and applications of the derivatization reagents, for the LC/MS/MS quantification of metabolites. This strategy has been used for the relative and absolute quantification of a variety of metabolites, and expansion of the coverage of metabolites.

Isobaric Tag-Based Protein Profiling across Eight Human Cell Lines Using High-Field Asymmetric Ion Mobility Spectrometry and Real-Time Database Searching.

A vast number of human cell lines are available for cell culture model-based studies, and as such the potential exists for discrepancies in findings due to cell line selection. To investigate this concept, the authors determine the relative protein abundance profiles of a panel of eight diverse, but commonly studied human cell lines. This panel includes HAP1, HEK293T, HeLa, HepG2, Jurkat, Panc1, SH-SY5Y, and SVGp12. A mass spectrometry-based proteomics workflow designed to enhance quantitative accuracy while maintaining analytical depth is used. To this end, this strategy leverages TMTpro16-based sample multiplexing, high-field asymmetric ion mobility spectrometry, and real-time database searching. The data show that the differences in the relative protein abundance profiles reflect cell line diversity. The authors also determine several hundred proteins to be highly enriched for a given cell line, and perform gene ontology and pathway analysis on these cell line-enriched proteins. An R Shiny application is designed to query protein abundance profiles and retrieve proteins with similar patterns. The workflows used herein can be applied to additional cell lines to aid cell line selection for addressing a given scientific inquiry or for improving an experimental design.

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.

Lowering Sample Requirements to Study Tyrosine Kinase Signaling Using Phosphoproteomics with the TMT Calibrator Approach.

Analysis of tyrosine kinase signaling is critical for the development of targeted cancer therapy. Currently, immunoprecipitation of phosphotyrosine (pY) peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to profile tyrosine kinase substrates. A typical protocol requests 10mg of total protein from ≈108 cells or 50-100mg of tissue. Large sample requirements can be cost prohibitive or not feasible for certain experiments. Sample multiplexing using chemical labeling reduces the protein amount required for each sample, and newer approaches use a material-rich reference channel as a calibrator to trigger detection and quantification for smaller samples. Here, it is demonstrated that the tandem mass tag (TMT) calibrator approach reduces the sample input for pY profiling tenfold (to ≈1mg total protein per sample from 107 cells grown in one plate), while maintaining the depth of pY proteome sampling and the biological content of the experiment. Data are available through PRIDE (PXD019764 for label-free and PXD018952 for TMT). This strategy opens more opportunities for pY profiling of large sample cohorts and samples with limited protein quantity such as immune cells, xenograft models, and human tumors.

Sample Multiplexing: Increased Throughput for Quantification of Total Testosterone in Serum by Liquid Chromatography-Tandem Mass Spectrometry.

For high-volume assays, optimizing throughput reduces test cost and turn-around time. One approach for liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays is sample multiplexing, wherein the analyte of interest is derivatized in different specimens with reagents of different molecular weight (differential mass tagging). Specimens can then be combined and simultaneously analyzed within a single injection to improve throughput. Here we developed and validated a quantitative, sample-multiplexed LC-MS/MS assay for serum total testosterone (TT) based on this approach. For the sample-multiplexed assay, calibrators, controls, and patient specimens were first extracted separately. After mass tagging with either methoxyamine or hydroxylamine, they were combined and injected into the LC-MS/MS system. To evaluate assay performance, we determined limit of quantification (LOQ), linearity, recovery, and imprecision. A method-comparison study was also performed, comparing the new assay with the standard LC-MS/MS assay in 1574 patient specimens. The method was linear from 2.5 to 2000 ng/dL, with accuracies from 93% to 104% for both derivatives. An LOQ of 1.0 ng/dL was achieved. Intra-assay and total CVs across 4 quality control concentrations were less than 10%. The assay demonstrated good agreement (Deming regression, 1.03x + 6.07) with the standard LC-MS/MS assay for the patient specimens tested (TT, 3 to 4862 ng/dL). Sample multiplexing by differential mass tagging of TT increases LC-MS/MS throughput 2-fold without compromising analytical accuracy and sensitivity.

Abstract 1352: Enhanced target capturing throughput and simplified workflow via innovative chemistry on hybrid capture blockers

Abstract Hybrid capture is a popular and well-accepted method to select targeted genomic regions for subsequent next generation sequencing (NGS). Despite its popularity in research and clinical diagnostics, hybrid capture NGS is now facing the challenge by the high throughput demands generated by Novaseq, Illumina's flagship platform that occupies the research and industry market rapidly in recent years. Novaseq create tetrabytes of sequencing data per run, allowing unprecedented level of sample multiplexing, especially for targeted NGS. Conventional blockers, whose sample index-matching region uses either degenerate bases or universal pairing bases, still need to match the length of the sample index for each individual library. When using NovaSeq, scientists need to combine huge number of samples using different types of index system in each single run. However, matching different hybridization blockers with corresponding index system is time consuming and error-prone. Here we introduce a novel design and chemistry to hybrid capture blockers to maximized the pooling capacity of hybrid capture to match the increasing need of sample throughput and multiplexing. Our new hybrid capture blockers are not only an essential component in the target enrichment workflow to block the permissive interactions between library adapters and target probes, which may lower the capturing efficiency and generate excessive data from the off-target sequence, but also significantly promotes the capturing capacity and efficiency, and simplifies the workflow. Our new blocker, GenFisher Universal Blockers, using our proprietary design and chemistry, create a novel solution to the conjugation between sample indexes on libraries and blockers, enabling blockers bind adapters regardless to the index length and sequence. In our experiment, NGS libraries are created with a commercially available kit and TruSeq style adapters that contain sample indexes with 3 different lengths: 6nt, 8nt and 10nt. Hybrid captures were then carried out with 8,000-probe GenFisher hybridization panel and kit and Universal Blockers for each individual libraries and the combined library with different indexes. Sequencing data showed that the on-target base percentage was consistently at &amp;lt;75% for all captures for both individual or combined multiplex libraries, whereas hybrid capture without blockers yielded &amp;gt;30% on-target rate. We also compared GenFisher Universal Blockers with the blockers from two other industrial leading providers, which are also claimed to be ‘universal'. Our data showed that GenFisher Universal Blockers was the only truly universal solution that worked with all 3 index lengths unbiasedly while the other two are still index length-sensitive and struggled with 10nt indexes library capturing (on-target ~50%, compared to &amp;lt;65% at 6/8nt). With its versatility, GenFisher Universal Blockers enables higher multiplexing capacity, as well as the freedom of index selection to hybrid capture NGS users. Citation Format: Jianpeng Wang, Long Fan, Lumeng Ye, Jiashi Wang, Irene Song, Cedric Wu. Enhanced target capturing throughput and simplified workflow via innovative chemistry on hybrid capture blockers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1352.

Sample multiplexing for targeted pathway proteomics in aging mice

Pathway proteomics strategies measure protein expression changes in specific cellular processes that carry out related functions. Using targeted tandem mass tags-based sample multiplexing, hundreds of proteins can be quantified across 10 or more samples simultaneously. To facilitate these highly complex experiments, we introduce a strategy that provides complete control over targeted sample multiplexing experiments, termed Tomahto, and present its implementation on the Orbitrap Tribrid mass spectrometer platform. Importantly, this software monitors via the external desktop computer to the data stream and inserts optimized MS2 and MS3 scans in real time based on an application programming interface with the mass spectrometer. Hundreds of proteins of interest from diverse biological samples can be targeted and accurately quantified in a sensitive and high-throughput fashion. It achieves sensitivity comparable to, if not better than, deep fractionation and requires minimal total sample input (∼10 µg). As a proof-of-principle experiment, we selected four pathways important in metabolism- and inflammation-related processes (260 proteins/520 peptides) and measured their abundance across 90 samples (nine tissues from five old and five young mice) to explore effects of aging. Tissue-specific aging is presented here and we highlight the role of inflammation- and metabolism-related processes in white adipose tissue. We validated our approach through comparison with a global proteome survey across the tissues, work that we also provide as a general resource for the community.