Sample Multiplexing as a Key Enabling Technology for Quantitative Proteomics

Summary: Sample multiplexing as a key enabling technology for quantitative proteomics Proteins are the biological effector molecules in cells and tissues—the molecules which carry out most catalytic, structural, and regulatory functions inside cells. Their rates of synthesis and degradation (and thus their abundance levels) are often controlled post-transcriptionally. Furthermore, the activities of proteins are frequently modulated by post-translational modifications (e.g., phosphorylation and ubiquitylation) and depend on other macromolecules, including DNA, RNA and proteins to interpret modifications and initiate new, condition-specific activities. Over 20 years of genomics and proteomics research has not succeeded in a general solution to the grand challenge of assigning function to the ~20,000 human genes. This reflects not only the magnitude of the challenge, but the lack of technologies that can be i) adapted to handle the throughput that complex experimental designs demand and ii) used to discover selective pharmacological tools to perturb and reveal a protein's function. In this R35 proposal, we address these two important research priorities in the field of proteomics. Mass spectrometry (MS)-based proteomics has evolved into an essential tool, allowing for both qualitative and quantitative analyses of biological systems. During the past 24 years, my lab has pioneered many approaches to study protein phosphorylation and ubiquitylation as well as directly profiling cell-state-specific protein expression differences by MS. In addition, our contributions to the sample multiplexing (isobaric tagging) field over the past 14 years are vast. Our current tandem mass tag (TMT) workflow supports up to 18 samples for simultaneous proteome-wide analysis. While sample multiplexing clearly increases throughput by more than an order of magnitude, in proteomics, there are other nonobvious key advantages. For example, an 18plex dataset acts as a remarkable closed system creating an N proteins x 18 conditions matrix with essentially no missing values, facilitating powerful statistical analyses. In addition, very complex experimental designs can be accommodated within the 18 samples to include biological replicates, time series, dose-response, rescue, positive and negative controls, etc. Thus, sample multiplexing in proteomics can arguably be thought of as the world's most powerful biological assay, accurately measuring the levels of up to 10,000 proteins across 18 conditions in less than one day. Over the next several years, we will improve isobaric tagging-based proteome analysis even more by i) increasing the multiplexing capacity to 35plex, ii) creating custom software to manage the entire proteome analysis in real time, iii) designing targeted sample multiplexing workflows for ultra-high throughput, and iv) creating and applying novel chemoproteomic workflows to identify chemical probes to illuminate protein function in an unbiased manner. Project Number: 1R35GM158136-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: STEVEN GYGI | Institution: HARVARD MEDICAL SCHOOL, BOSTON, MA | Award Amount: $2,527,500 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MCST-Q (50)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11089692