Rapid communications in mass spectrometry : RCM | 2019
Microwave-assisted acid hydrolysis for whole bone proteomics and paleoproteomics.
Abstract
RATIONALE\nWhole bone proteomic analyses rely on lengthy sample preparation including demineralization and digestion to break bone down into peptides to recover using mass spectrometry. However, microwave-assisted acid hydrolysis, a technique used in proteomic analyses on other soft tissues and cells, will combine both demineralization and digestion and only take minutes.\n\n\nMETHODS\nTo test microwave-assisted hydrolysis on whole moose bone, we microwaved 5 concentrations of acetic and formic acid (15%, 12.5%, 10%, 7.5% and 5%) for three times (10, 20 and 30 minutes) at 140o C using an ETHOS UP High Performance Microwave Digestion System. Peptides were injected and separated using Thermo BioBasic C18 columns and detected on an LTQ Orbitrap Velos mass spectrometer. We searched the raw data on PEAKS 8.5 against the white-tailed deer database.\n\n\nRESULTS\nFormic acid hydrolysis led to the most complete digestion, and therefore the highest number of PSMs, more protein groups and better sequence coverage for collagenous proteins. However, in the formic acid samples there is a tradeoff with digestion completeness and a higher incidence of in vitro modifications (i.e., formylation) that are not induced using acetic acid. Acetic acid has greater cleavage specificity and higher sequence coverage for non-collagenous proteins.\n\n\nCONCLUSIONS\nDepending on the goals of analysis, there are benefits and drawbacks to using both acetic and formic acid. Overall, microwave-assisted acid hydrolysis was successful in demineralizing and digesting bone fragments to considerably speed up the preparation for bottom-up proteomic analysis.