HUPO 2024 Poster Information
Poster Abstracts
P01.37 | From Volcanoes to the Bench: Advantages of Novel Hyperthermoacidic Archaeal Proteases for Proteomics Workflows
Monday March 11, 13:30-15:00
Steven M. Yannone (1), Maxwell McCabe (2), Varun Gejji (1), Adam Barnebey (1), G. Suizdak (3), Lihn T. Hoang (3), Anthony J Saviola (2), Kirk Hansen (2)
Here we introduce hyperthermoacidic archaeal proteases (HTA-Proteases©) isolated from organisms that thrive in nearly boiling acidic volcanic springs around the world. HTA-Proteases function optimally at 70- 90°C and pH of 2-4 with rapid digestion kinetics. The extreme heat and acidity of HTA-Protease reactions denatures sample proteins and obviates the use of chaotropes and facilitates one-step/five-minute sample preparation protocol without sample manipulation, dilution or cleanup thereby reducing/eliminating sample loss. We also find that HTA-Proteases have novel cleavage specificity, show no autolysis, function in dilute formic acid, and ship and store in aqueous buffer at ambient temperature for over two years. We find that brief one-step HTA-Protease protocols significantly increase proteome and protein sequence coverage with datasets orthogonal to trypsin. Importantly, HTA-Protease digests show a bias for identification of ribonucleoproteins, histones, mitochondrial, and membrane proteins. HTA-Protease one-step protocols in dilute formic acid are of possible advantage for many areas of proteomic research.
(1) CinderBio, San Leandro, CA)
(2) Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Denver, CO
(3) The Scripps Research Institute, La Jolla, CA
P03.08 | Near Real-Time Monitoring of Blood Biomarkers with “Rapid Proteomics”
Tuesday March 12, 13:30-15:00
Steven M. Yannone (1), Jennifer Van Eyk (2), Simion Kreimer (2)
Multi-hour tryptic proteolysis disqualifies conventional bottom-up proteomics from diagnosis of acute clinical conditions because a fast turn-around is needed to monitor and mitigate rapidly progressing physiological reactions. The Krakatoa protease manufactured by CinderBio proteolyzes samples at pH 3 and 80-90 C in just 10-30 minutes. “Rapid Proteomics” leverages quick sample preparation and high throughput LC-MS for a turn-around of 30-60 minutes from blood collection to actionable data to quantify circulating biomarkers in near real-time. The Krakatoa protease most frequently cleaves Leucine, Phenylalanine, and Glutamic acid but with limited specificity and many missed cleavages. Nonetheless qualification of a 20-minute proteolysis of 1 microliter of venous blood from a finger prick with a 20-minute data independent acquisition (DIA) LC-MS analysis found that over 160 circulating proteins were reproducibly quantified. Over 100 proteins were quantified at CV <30% inter-day imprecision based on 5 replicates prepared on 3 different days (n = 15) in blood from 3 healthy donors. Over 120 proteins had a CV <20% intraday imprecision across all 3 days (5 replicates in one batch). Optimization of the proteolysis duration spanning 10 to 60 minutes found that proteins were digested after just 15 minutes while the larger peptide were cleaved after 60 minutes. This indicates that the proteolysis strongly favors large substrates and circulating bioactive peptide hormones like Angiotensin I and II and Bradykinin are retained and protected from endogenous proteases by the acidity of the digestion buffer. An 8-minute targeted LC-MS method was developed to quantify bradykinin and angiotensin variants after a 20-minute proteolysis of 1 microliter of venous blood. This inaugural Rapid Proteomics method allows measurement of these critical hormones and several additional proteins at 8-minute intervals with a 30-minute delay to monitor Phase 1 drug responses, allergic challenge reactions, and other other applications.
(1) CinderBio, San Leandro, CA)
(2) Cedars-Sinai Medical Center, Los Angeles, CA
