Joining Two Perspectives of the Proteome with the Trypsin+HTA Protease Workflow
Proteolysis can be completed in a single-step, rapid (5-30 minutes) reaction using hyperthermoacidic archaeal (HTA) proteases. Protocol simplicity and stability of HTA proteases in ambient storage conditions removes many logistical barriers to widespread deployment of proteomic analysis. HTA protease cleavage selectivity is complicated but quantitatively reproducible and generates peptides that…
The trypsin+HTA protease workflow combines two protease techniques to enhance proteomic analysis. Hyperthermoacidic archaeal (HTA) proteases enable rapid (5-30 minutes) single-step protein digestion, while trypsin provides established selectivity. The two peptide sets generated by the process have minimal overlap, offering complementary coverage of amino acid sequences and post-translational modifications (PTMs).
Mixing HTA protease peptides with trypsin digestion in liquid chromatography-mass spectrometry (LC-MS) analysis improves protein identification by 24% on average, particularly in plasma samples. This combination identified 216 additional non-artifact modifications compared to trypsin-only analysis. The method is reproducible and does not increase sample preparation or analysis time.
A quantification model selects the protease with the most complete data for each protein and uses the second protease to infer missing values, providing more accurate cross-protease imputations than alternative methods. Biomarkers derived from HTA protease peptides can be analyzed using HTA protease-only workflows, facilitating translation of discoveries into practical applications.
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