Museum Conservation Institute, Smithsonian Institution, Suitland, MD, 20746, USA.
Rapid Commun Mass Spectrom. 2020 Jan 30;34(2):e8568. doi: 10.1002/rcm.8568.
Whole-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 of other soft tissues and cells, will combine both demineralization and digestion and only take minutes.
To test microwave-assisted hydrolysis on whole moose bone, we microwaved five concentrations of acetic and formic acids (15%, 12.5%, 10%, 7.5% and 5%) for three times (10, 20 and 30 min) at 140°C using an ETHOS UP high performance microwave digestion system. Peptides were injected and separated using Thermo BioBasic C18 columns and detected with an LTQ Orbitrap Velos mass spectrometer. We searched the raw data on PEAKS 8.5 against the white-tailed deer database.
Formic acid hydrolysis led to the most complete digestion, and therefore the highest number of peptide spectrum matches, more protein groups and better sequence coverage for collagenous proteins. However, for 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.
Depending on the goals of analysis, there are benefits and drawbacks to using both acetic acid 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 proteomics analysis.
全骨蛋白质组学分析依赖于冗长的样品制备，包括脱矿和消化，将骨骼分解成肽，然后使用质谱法回收。然而，微波辅助酸水解是一种用于其他软组织和细胞蛋白质组学分析的技术，它将同时进行脱矿和消化，仅需几分钟。
为了在整个驼鹿骨上测试微波辅助水解，我们使用 ETHOS UP 高性能微波消解系统，在 140°C 下用 15%、12.5%、10%、7.5%和 5%的醋酸和甲酸各微波处理三次（每次 10、20 和 30 分钟）。使用 Thermo BioBasic C18 柱注入和分离肽，并使用 LTQ Orbitrap Velos 质谱仪进行检测。我们在 PEAKS 8.5 上针对白尾鹿数据库搜索原始数据。
甲酸水解导致最完全的消化，因此肽谱匹配、更多的蛋白质组和更好的胶原蛋白序列覆盖率最高。然而，对于甲酸样品，消化完整性与更高的体外修饰（即甲酰化）发生率之间存在权衡，而使用乙酸则不会诱导这些修饰。乙酸对非胶原蛋白具有更强的裂解特异性和更高的序列覆盖率。
根据分析的目标，使用乙酸和甲酸都有其优缺点。总体而言，微波辅助酸水解成功地对骨碎片进行脱矿和消化，大大加快了用于自上而下蛋白质组学分析的准备工作。
