Online Microwave D-Cleavage LC-ESI-MS/MS of Intact Proteins: Site-Specific Cleavages at Aspartic Acid Residues and Disulfide Bonds

Abstract

An online nonenzymatic digestion method utilizing a microwave-heated flow cell and mild acid hydrolysis at aspartic acid (D) for rapid protein identification is described. This methodology, here termed microwave D-cleavage, was tested with proteins ranging in size from 5 kDa (insulin) to 67 kDa (bovine serum albumin) and a bacterial cell lysate ( Escherichia coli). A microwave flow cell consisting of a 5 microL total volume reaction loop connected to a sealed reaction vessel was introduced into a research grade microwave oven. With this dynamic arrangement, the injected sample was subjected to microwave radiation as it flowed through the reaction loop and was digested in less than 5 min. Different digestion times can be achieved by varying the sample flow rate and/or length of the loop inside the microwave flow cell. The microwave flow cell can be operated individually with the output being collected for matrix assisted laser ionization/desorption (MALDI) mass spectrometry (MS) or connected online for liquid chromatography (LC) electrospray ionization (ESI)-MS. In the latter configuration, the microwave flow cell eluates containing digestion products were transferred online to a reversed phase liquid chromatography column for direct ESI-MS and ESI-MS/MS analyses (specifically, Collision Induced Dissociation, CID). Concurrently with the microwave D-cleavage step, disulfide bond reduction/cleavage was achieved by the coinjection of dithiothreitol (DTT) with the sample prior to online microwave heating and online LC-MS analysis and so eliminating the need for alkylation of the reduced protein. All protein standards, protein mixtures, and proteins in a bacterial cell lysate analyzed by this new online methodology were successfully identified via a SEQUEST database search of fragment ion mass spectra. Overall, online protein digestion and identification was achieved in less than 40 min total analysis time, including the chromatographic step.

Rapid Online Non-Enzymatic Protein Digestion Analysis with High Pressure Superheated ESI-MS

“…The mass spectra of ubiquitin obtained at 180°C are shown in Figure2a. Except for the peptide fragment (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), all other hydrolysis products are due to the cleavage at the Asp sites. The result with acetic acid (Figure2b) shows the hydrolysis at exactly the same cleavage sites, but in contrast to formic acid, there is also a presence of dehydrated species (e.g., (M+nH−H 2 O) n+ .…”

“…Polar solvents like aqueous solution can be efficiently and uniformly heated by microwave excitation at 2.45GHz, and there is rich literature on the microwave-assisted chemical digestion [6,7,[9][10][11][12][13][14]. The digestion process that typically requires several hours to complete can be accelerated to about 5-10min by incubating the sample in the microwave oven.…”

Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions

“…This approach exploits unique microwave properties to accelerate the enzymatic digestion. In recent years, microwaveassisted proteolytic digestion procedures, including tryptic [6][7][8] and acid-mediated proteolysis [9][10][11][12], have enabled samples to be prepared more rapidly for bottom-up analysis.…”

When API Mass Spectrometry Meets Super Atmospheric Pressure Ion Sources

“…[51][52][53][54][55] is type of chemical digestion has a selective cleavage at the aspartate (Asp, D) residue. Microwave oven is usually used for this purpose and ESI-MS is done in an o -line basis.…”