Abstract
While the bottom-up protein analysis serves as a mainstream method for biological studies, its efficiency is limited by the time-consuming process for enzymatic digestion or hydrolysis as well as the post-digestion treatment prior to mass spectrometry analysis. In this work, we developed an enzyme-free microreaction system for fast and selective hydrolysis of proteins, and a direct analysis of the protein digests was achieved by nanoESI (electrospray ionization) mass spectrometry. Using the microreactor, proteins in aqueous solution could be selectively hydrolyzed at the aspartyl sites within 2 min at high temperatures (∼150 °C). Being free of salts, the protein digest solution could be directly analyzed using a mass spectrometer with nanoESI without further purification or post-digestion treatment. This method has been validated for the analysis of a variety of proteins with molecular weights ranging from 8.5 to 67 kDa. With introduction of a reducing agent into the protein solutions, fast cleavage of disulfide bonds was also achieved along with high-temperature hydrolysis, allowing for fast analysis of large proteins such as bovine serum albumin. The high-temperature microreaction system was also used with a miniature mass spectrometer for the determination of highly specific peptides from Mycobacterium tuberculosis antigens, showing its potential for point-of-care analysis of protein biomarkers.
Highlights
Mass spectrometry (MS) has become a powerful tool for identi cation of protein primary sequences, post-translational modi cations (PTMs) and protein–protein interactions; it enables determination of various cellular functions at the protein level.[1]
We developed an enzyme-free microreaction system for fast and selective hydrolysis of proteins, and a direct analysis of the protein digests was achieved by nanoESI mass spectrometry
A protein solution prepared with water and weak acid was loaded into the PFA tubing using a syringe; both ends of the tubing were sealed by using two removable stoppers and the heating was performed for less than 2 min; a er cooling in air for 30–60 s, the protein solution was transferred into a glass capillary (i.d. 0.8 mm) with a pulled tip in an offline fashion for subsequent nanoESI-MS analysis
Summary
Mass spectrometry (MS) has become a powerful tool for identi cation of protein primary sequences, post-translational modi cations (PTMs) and protein–protein interactions; it enables determination of various cellular functions at the protein level.[1]. Proteins in aqueous solution could be selectively hydrolyzed at the aspartyl sites within 2 min at high temperatures ($150 C).
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