TASK 4 GENOME-WIDE PROFILING OF PROTEINS Overall objective and rationale The objective of Task 4 is to provide high quality, robust, deep proteomics data for the TOPMed consortium. Proteomics data are of high value: a) the levels of many proteins do not often correspond to RNA levels and proteins may be closer to phenotypes than RNA measurements1. b) Proteins are often the targets of drugs and thus valuable for therapeutics c) Protein activities are often modified by posttranslational modification which can be directly measured. Proteins exhibit a large dynamic range (up to 10E12) in plasma making comprehensive analysis challenging2. Recent advances have enabled deep protein profiling, which fall into two general approaches: measurements using capture agents and those using mass spectroscopy (MS) (Table 1). Each measures relative levels of proteins, rather than absolute quantification. The dominant capture strategies are Olink3 and Somascan4. Although Somacan profiles more proteins (presently 11,000 vs 5,400 for Olink) for approximately the same cost, Olink uses paired antibodies against different epitopes which is expected to greatly increase specificity - a major concern with capture agents. It is worth noting that a large study comparing both technologies found a correlation of 0.46, indicating significant differences in the measurements, particularly for the low abundance proteins. MS is a complementary method. It is typically used in a bottom-up strategy where cell lysates are digested with trypsin and the resulting peptides analyzed using LC/MS/MS and sometimes LC/MS/MS/MS5. The latter identifies fewer proteins but provides more accurate quantification. Peptide identification is performed by matching to protein databases. Plasma is often dominated by several highly abundant signals which makes detection of low abundance proteins difficult. Two methods to circumvent the problem, at least in part, are: 1) Affinity removal of the 14 most abundant proteins for plasma enabling the identification of the remaining lower abundance proteins and 2) SEER technology which uses affinity absorption to capture complex protein space and reduce analysis of the most abundant proteins. Capture methods and mass spectrometry methods are complementary. Capture methods profile high value targets such as those implicated in cardiovascular disease and inflammation including many low abundance proteins such as cytokines. Mass spectrometry, on the other hand, typically identifies the most abundant ~8000 proteins which often reflect cell identity and relevant biological processes. Thus if sufficient funds exist, using both technologies obtains a very broad range of proteins. Presently most major projects (DECODE, 100K UK) use Olink because it has higher throughput, however, the latest Astral instrument from Thermo has greatly accelerated mass spectrometry profiling rate up to 300 samples/day with deep proteome coverage. Task Order 5 Single-Cell Omics Overall appr...