Benefit of microwave‐assisted acid hydrolysis of proteins for mass spectrometric profiling of the human heart tissue proteome

Gebremedhin, Mulu; Zhong, Hongying; Wang, Shaohua; Weinfeld, Michael; Li, Liang

doi:10.1002/rcm.3125

Cited by 7 publications

(2 citation statements)

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“…In addition, using a weak acid, such as 25% trifluoroacetic acid (TFA), terminal and internal peptides can be generated by MAAH for shotgun proteome analysis [2]. This method is particularly useful for analyzing insoluble proteins, such as those found in tissue samples [4,9], as the microwave technique can assist in protein solubilization during MAAH, and proteins not efficiently digested by using conventional enzymes [11]. In addition, microwave hydrolysis can accelerate the hydrolysis process and generate more reproducible results, compared with heating alone, and it can be more readily applied to membrane proteins as heating alone causes precipitation of membrane proteins in acids which prevent them from further degradation [1,2].…”

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confidence: 99%

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry

Reiz

2010

J. Am. Soc. Mass Spectrom.

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Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein. (J Am Soc Mass Spectrom 2010, 21, 1596 -1605

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Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry

Reiz

2010

J. Am. Soc. Mass Spectrom.

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“…This is particularly true for hydrophobic membrane proteins [14,21]. Furthermore, a protein sam-ple does not need to be completely dissolved in TFA for MAAH-merely suspending the protein sample in TFA is sufficient for acid hydrolysis [21,37]. However, in the traditional microwave experiment using a household microwave oven [13,14,38], a sample vial containing a low volume of solution (40 L) only absorbs a small fraction of the microwave energy.…”

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confidence: 99%

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

Wang

2010

J. Am. Soc. Mass Spectrom.

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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...

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Microwave Technology to Accelerate Protein Analysis

Mirza

Pramanik

Bose

2011

Protein and Peptide Mass Spectrometry in Drug Discovery

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Benefit of microwave‐assisted acid hydrolysis of proteins for mass spectrometric profiling of the human heart tissue proteome

Cited by 7 publications

References 31 publications

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry

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

Microwave Technology to Accelerate Protein Analysis

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