Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Abstract

Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 g of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/ l or 37 fmol/ l. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein. (J Am Soc Mass Spectrom 2005, 16, 471-481)

Microwave‐assisted proteomics

“…In a subsequent publication by Zhong, Marcus, and Li (2005), a method for microwave-assisted acid cleavage of proteins at acid labile sites was described (refer to Section V.B). One of the caveats of their preliminary acid hydrolysis conditions was that MAAH in the presence of 1.5 M TFA for a few minutes resulted in predominantly cleavage of only the termini.…”

“…One of the caveats of their preliminary acid hydrolysis conditions was that MAAH in the presence of 1.5 M TFA for a few minutes resulted in predominantly cleavage of only the termini. This cleavage pattern provided an alternative method to sequence the termini of proteins to the previously described HCl ladder hydrolysis (Zhong, Marcus, & Li, 2005).…”

“…Just a few years later, microwave technology began to emerge in protocols of a more bio-analytical nature. Such preliminary techniques included the exposure of antigens for immunohistochemical staining in embedded tissue samples (Patterson & Bulard, 1980;Hafajee & Leong, 2004;Emersen et al, 2006;Temel et al, 2006), increased acid hydrolysis of proteins for amino acid analysis (Gilman & Woodward, 1990;Chiou & Wang, 1990;Chen, Chiou, & Wang, 1991;Davidson, 1996), increased catalysis of enzymatic and chemical cleavages for peptide mapping (Zhong, Marcus, & Li, 2005;Hua, Low, & Sze, 2005), and increased removal of PTMs for improved protein characterization (Lee et al, 2005a,b;Sandoval et al, 2007).…”

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.…”

Acid hydrolysis of proteins in matrix assisted laser desorption ionization matrices

“…Similar to improvements in trypsin digestion, microwave-assisted acid hydrolysis (MAAH) has been shown to speed the formation of peptide fragments when compared with the original in solution method [9]. MAAH methods have also been demonstrated for 25% trifluoroacetic acid [10] and 12.5% acetic acid [11,12]. These methods are compatible with downstream liquid chromatography and mass spectrometry, and they have been applied to the identification of bacterial spores [13] and viruses [14], as well as proteome analysis of yeast ribosomes [11,12] and zebrafish liver [15].…”