Reproducible microwave-assisted acid hydrolysis of proteins using a household microwave oven and its combination with LC-ESI MS/MS for mapping protein sequences and modifications

Abstract

A new set-up for microwave-assisted acid hydrolysis (MAAH) with high efficiency and reproducibility to degrade proteins into peptides for mass spectrometry analysis is described. It is based on the use of an inexpensive domestic microwave oven and can be used for low volume protein solution digestion. This set-up has been combined with liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI QTOF MS) for mapping protein sequences and characterizing phosphoproteins. It is demonstrated that for bovine serum albumin (BSA), with a molecular mass of about 67,000 Da, 1292 peptides (669 unique sequences) can be detected from a 2 g hydrolysate generated by trifluoroacetic acid (TFA) MAAH. These peptides cover the entire protein sequence, allowing the identification of an amino acid substitution in a natural variant of BSA. It is shown that for a simple phosphoprotein containing one phosphoform, ␤-casein, direct analysis of the hydrolysate generates a comprehensive peptide map that can be used to identify all five known phosphorylation sites. For characterizing a complex phosphoprotein consisting of different phosphoforms with varying numbers of phosphate groups and/or phosphorylation sites, such as bovine ␣ S1 -casein, immobilized metal-ion affinity chromatography (IMAC) is used to enrich the phosphopeptides from the hydrolysate, followed by LC-ESI MS analysis. The MS/MS data generated from the initial hydrolysate and the phosphopeptide-enriched fraction, in combination with MS analysis of the intact protein sample, allow us to reveal the presence of three different phosphoforms of bovine ␣ S1 -casein and assign the phosphorylation sites to each phosphoform with high confidence. (J Am Soc Mass Spectrom 2010, 21, 1573-1587) © 2010 American Society for Mass Spectrometry P rotein sequence mapping is commonly used to study post-translational modifications of a protein or amino acid substitutions from point mutations in the genome. Ideally, the entire amino acid sequence of a protein should be mapped to pinpoint where a modified amino acid or a substitution is located. Mass spectrometry (MS) has become an indispensable tool for protein sequence mapping [1,2]. This is usually done by using a top-down or a bottom-up proteomic approach [1][2][3][4][5][6][7][8][9][10][11][12]. In the top-down method, a protein ion is dissociated in a tandem mass spectrometer (MS/MS) and the fragment ions generated are interpreted to generate a stretch of amino acid sequence information. Sequence coverage by this method is dependent on the nature of the protein, ranging from a few residues to a full sequence [12]. In general, full sequence information is difficult to obtain for a protein with molecular mass of Ͼ20,000 Da [12]. The bottom-up approach is a robust method for protein identification based on sequencing by MS/MS one or more peptides generated by chemical or enzymatic degradation of a protein. For a protein digest, such as that produced by trypsin digestion, one or a few peptides are sequenced, r...

Ser71 of αS1‐Casein is Phosphorylated in Breast Milk—Evidence from Targeted Mass Analysis

“…However, it also noted a dependency from the peptide sequence so that the implications for the behavior of the intact protein remain open. Using hydrolysis and LC‐MS/MS Wang and Li could not confidently assign the phosphorylation sites in the sequence part 79 SISSS 83 . A study based on β‐elimination and cation‐exchange chromatography claimed to have detected five phosphosites in the serine‐rich region but only showed MS/MS data for spiked reference peptides .…”

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.…”

Mass spectrometry-based proteomics as a tool to identify biological matrices in forensic science

“…To further optimize this approach for implementation, automated digestion can be used to diminish the workload: shorter digestion times have already been described (2.5 s–7.5 min) and will dramatically decrease the sample preparation time [32, 33]. Data analysis can be simplified by developing a selected reaction monitoring (SRM) workflow on proteotypic peptides [34].…”