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Ultrafast enzymatic digestion of proteins by microdroplet mass spectrometry
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Ultrafast enzymatic digestion of proteins by microdroplet mass spectrometry
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Journal Article
Ultrafast enzymatic digestion of proteins by microdroplet mass spectrometry
2020
Overview
Enzymatic digestion for protein sequencing usually requires much time, and does not always result in high sequence coverage. Here we report the use of aqueous microdroplets to accelerate enzymatic reactions and, in particular, to improve protein sequencing. When a room temperature aqueous solution containing 10 µM myoglobin and 5 µg mL
−1
trypsin is electrosonically sprayed (−3 kV) from a homemade setup to produce tiny (∼9 µm) microdroplets, we obtain 100% sequence coverage in less than 1 ms of digestion time, in sharp contrast to 60% coverage achieved by incubating the same solution at 37 °C for 14 h followed by analysis with a commercial electrospray ionization source that produces larger (∼60 µm) droplets. We also confirm the sequence of the therapeutic antibody trastuzumab (∼148 kDa), with a sequence coverage of 100% for light chains and 85% for heavy chains, demonstrating the practical utility of microdroplets in drug development.
Mass spectrometry (MS)-based protein sequencing usually relies on in-solution proteolytic digestion, which is time-consuming and inefficient for certain proteins. Here, the authors achieve full protein sequence coverage in less than 1 ms by subjecting protein-protease mixtures to electrosonic spray ionization-MS.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 639/638
/ Adrenocorticotropic Hormone - analysis
/ Adrenocorticotropic Hormone - metabolism
/ Chains
/ Enzymes, Immobilized - chemistry
/ Humanities and Social Sciences
/ Humans
/ Ions
/ Proteins
/ Science
/ Sequence Analysis, Protein - methods
/ Spectrometry, Mass, Electrospray Ionization - methods
/ Trypsin
