From Volcanos to the Bench: Advantages of Novel Hyperthermoacidic Archaeal Proteases for Proteomics Workflows

ABSTRACT: Here we introduce novel hyperthermoacidic archaeal proteases (HTA-Proteases) isolated from organisms that thrive in nearly boiling acidic volcanic springs and investigate their use for bottom-up proteomic experiments. We find that HTA-Proteases have novel cleavage specificities, show no autolysis, function in dilute formic acid, and store at ambient temperature for years. HTA-Proteases function optimally at 70-90°C and pH of 2-4 with rapid digestion kinetics. The extreme reaction conditions actively denature sample proteins, obviate the use of chaotropes, and allow for a one-step/five-minute sample preparation protocol without sample manipulation, dilution, or additional cleanup. 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 markedly increase coverage and identifications for ribonucleoproteins, histones, and mitochondrial membrane proteins as compared to tryptic digests. In addition to increased coverage in these classes, HTA-Proteases and simplified one-step protocols are expected to reduce technical variability and advance the fields of clinical and high-throughput proteomics. This work reveals significant utility of heretofore unavailable HTA-Proteases for proteomic workflows. We discuss some of the potential for these remarkable enzymes to empower new proteomics methods, approaches, and biological insights.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive HF, LTQ XL

ORGANISM(S): Bos Taurus (bovine) Homo Sapiens (human) Escherichia Coli Sus Scrofa Domesticus (domestic Pig) Mus Musculus (mouse)

TISSUE(S): Permanent Cell Line Cell, Whole Body, Cell Culture, Blood

SUBMITTER: Maxwell McCabe  

LAB HEAD: Kirk Hansen

PROVIDER: PXD041226 | Pride | 2023-10-18

REPOSITORIES: Pride