Additional possible tools for identification of proteins on one‐ or two‐dimensional electrophoresis

Abstract

Additional, essentially chemical, identification methods of proteins in polyacrylamide gel electrophoresis are described. Two cleavages of peptide bonds were used at the C-side of aspartic acid with a 0.2% pentafluoropropionic acid (PFPA) aqueous vapor at 90 degrees C for 4-16 h, and the N-side of serine/threonine with an S-ethyl trifluorothioacetate vapor at 50 degrees C for 6-24 h. The products were analyzed by mass spectrometry-peptide mass fingerprinting. A new type of C-terminal sequencing at multisites of protein was introduced. An aqueous vapor of 90% PFPA at 90 degrees C for 2-16 h provided cleavages at the C-side of aspartic acid and the N-side of serine/threonine and simultaneous successive truncation at the C-termini of the cleaved fragments. The product resulted in C-terminal sequences at multisites in proteins by mass spectrometric analysis. The following chemical deblocking methods were used. Anhydrous hydrazine vapor at -5 degrees C for 8 h deblocked the N-formyl group, and the vapor at 20 degrees C for 4 h deblocked pyrrolidone carboxylate. N-acetylserine/threonine was deblocked by aqueous vapor of 75% PFPA at 50 degrees C for 1 h, followed by reaction with p-sulfophenylisothiocyanate at pH 6.0. These methods were applied to a variety of protein spots on polyacrylamide gels. A new stepwise C-terminal sequencing of protein from polyacrylamide gels is also described.

Sequence information of peptides and proteins with in-source decay in matrix assisted laser desorption/ionization-time of flight-mass spectrometry

“…Mass spectrometry (MS) has been recognized as a powerful tool for the proteome or for proteomics because of the prompt acquisition performance of M r , peptide mass fingerprints, and amino acid sequencing. The use of newly developed gentle ionization methods such as electrospray ionization (ESI) [2] and matrix assisted laser desorption/ionization (MALDI) [3] allows us to obtain the molecular mass and peptide mass fingerprints of analyte proteins at low picomole levels [4,5]. Further, MS is a promising method for amino acid sequencing of protein fragments obtained by enzymatic digestion or chemical degradation [6].…”

Application of chemical selective cleavage methods to analyze post-translational modification in proteins

“…5c). No reaction was also observed for these peptides by the Asn-C cleavage reaction [7]. This desulfonylation also gives -80 m/z which is similar to dephosphorylation.…”

“…These amino acids are involved in acetylation, phosphorylation and glycosylation sites of protein. The second reaction is specific cleavage at the C-side of aspartyl peptide bonds with a vapor generated from 0.2% C 2 F 5 CO 2 H aqueous solution, containing 5% DTT, at 907C for 4-8 h [2,7]. The Asp residue covers the deamidation site of an Asn residue.…”

“…Vacuum dried peptide or protein (5-200 pmol), or the protein (5-100 pmol) on electroblotted membrane was placed in the small tube and 100 mL of 0.2% C 2 F 5 CO 2 H aqueous solution containing 5% DTT was placed in the large tube, and the reaction was carried out in the double tube system at 907C for 8 h [2,7]. Asp-Pro peptide bond was cleaved with 0.2% C 2 F 5 CO 2 H at 607C for 8 h. 0.2% C 2 F 5 CO 2 H. 10 mM DTT aqueous solution (50 mL) was directly reacted with the vacuum dried sample (5-100 pmol) under the same conditions.…”

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

“…Protein degradation is commonly carried out by using an enzyme such as trypsin. Chemical reagents such as CNBr 1 and acids [2][3][4][5][6][7][8][9][10][11] have also been used for protein degradation. Recent work indicates that both trypsin digestion and acid hydrolysis can be accelerated by using microwave irradiation.…”