Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis

Heß, Daniel; Covey, Tom; Winz, Robert A.; Brownsey, Roger W.; Aebersold, Ruedi

doi:10.1002/pro.5560020817

Cited by 92 publications

(51 citation statements)

References 32 publications

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“…The identification of a recombinant protein with differing modifications at the N-terminus has been observed previously by mass spectrometry (Dizhoor et al, 1992;Hess et al, 1993;Bradshaw & Stewart, 1994). However, the isolation and biochemical utilization of a formylated and deformylated species, as far as we know, is S.P.…”

mentioning

confidence: 81%

Identification and structural influence of a differentially modified N‐terminal methionine in human S l00b

1997

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Abstract:The calcium-binding protein SlOOb is a homodimer comprised of two identical 91-residue &subunits. Recombinant SlOOb is a heterogeneous protein, although the basis of this heterogeneity has not been established. We have used mass spectrometry and NMR spectroscopy to determine that heterogeneity in SlOOb arises from a mixture of formyl-SlOOb and desformylSlOOb when expressed in Escherichia coli. Reversed-phase HPLC purification of these two forms of SlOOb has allowed the differences in N-terminal composition to be used as a probe for tertiary contacts in the protein. The presence or absence of the N-terminal formyl group affected the chemical shifts of sequence neighboring residues and those in the linker of the protein (residues 40-43), indicating that these two regions are close in space.Keywords: calcium-binding protein; mass spectrometry; N-terminal formylation; protein heterogeneity; S 100 protein Posttranslational modification of the N-terminus of a protein in the cell is an important process. Typically in eukaryotic systems, the initiating residue, methionine, is cleaved enzymatically by methionine aminopeptidase to yield the mature protein, a process dependent on the nature of the penultimate N-terminal residue (Hire1 et al., 1989). In addition, the protein may be modified at the N-terminus to yield a variety of posttranslational modifications, including acetylation and myristoylation ( M h & Bradshaw, 1989;McIIhinney, 1990). Some of these modifications are necessary for targeting of the protein to the cell membrane. In Escherichia coli, frequently used for expressing recombinant proteins, the situation becomes more complex. Rather than acetylation, proteins are synthesized with an N-formyl methionine, which can be processed by a deformylase (Adams, 1968;Maze1 et al., 1994) and an aminopeptidase (Ben-Bassat et al., 1987). This can result in an N-terminus consisting of several different chemical species, including the formylMet, Met, and desMet forms.

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confidence: 81%

Identification and structural influence of a differentially modified N‐terminal methionine in human S l00b

1997

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“…1A). The labeled peptides were identified in a second run using tandem mass spectrometry in neutral loss mode, in which the ions are subjected to limited fragmentation by an inert gas in a collision cell (13,18,25). The ester bond between the inhibitor and the peptide is one of the more labile bonds present and would be expected to readily undergo homolytic cleavage resulting in loss of a neutral sugar.…”

Section: Identification Of the Labeled Active Site Peptides By Massmentioning

confidence: 99%

Unequivocal Identification of Asp-214 as the Catalytic Nucleophile of Saccharomyces cerevisiae α-Glucosidase Using 5-Fluoro Glycosyl Fluorides

McCarter

Withers²

1996

Journal of Biological Chemistry

130

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Yeast ␣-glucosidase is a member of a sequence-related family of ␣-glycosidases (Family 13) that includes important digestive ␣-amylases and ␣-glucosidases. These enzymes catalyze the hydrolysis of ␣-linked oligosaccharides by a two-step mechanism involving a glycosylenzyme intermediate. This Glycosyl hydrolases of Family 13 (the ␣-amylase family (1, 2)), which include ␣-amylases and some ␣-glucosidases and oligo-1,6-glucosidases, are important enzymes involved in the digestion of starch and other ␣-linked oligosaccharides in bacteria, plants, and animals. These glycosyl hydrolases cleave the glycosidic bond with net retention of configuration, thus are retaining enzymes that function via a two-step, double displacement mechanism. The first step involves the attack of an enzymic nucleophile at the anomeric center of the sugar, along with general acid catalysis to facilitate loss of the leaving group, leading to the formation of a ␤-D-glycosyl enzyme intermediate. This intermediate can then undergo hydrolysis in a second step, with general base catalysis to facilitate attack of water, resulting in cleavage of the glycosidic bond with net retention of anomeric configuration (see Scheme 2). The active site of these enzymes is known to include a trio of conserved carboxylic acids (3-7). Presumably, one residue functions as the catalytic nucleophile, one as a general acid/base, and the third possibly affords additional stabilization of developing positive charge or modulates the ionization behavior of the other catalytic residues.Considerable progress has been made toward elucidating the roles of these carboxylates in Family 13 enzymes, involving kinetic analyses of site-specific mutants (Ref. 8 and references therein) and x-ray crystallography (3-6). Labeling studies have also been useful. For example, an aspartate that functions as a catalytic nucleophile in a sucrose, ␣-glucan glycosyltransferase, has been identified in Streptococcus ␣-glucosyltransferase by denaturation trapping using radiolabeled sucrose (9). Similarly, in a second group of ␣-glycosyl hydrolases (Family 31), Asp-505 and Asp-1394 have been identified as active site residues in each of the two homologous active sites in the sucraseisomaltase complex by affinity labeling with conduritol B epoxide and have been suggested to be the catalytic nucleophiles (10). The asparagine mutant of the equivalent Asp-518 in human lysosomal ␣-glucosidase exhibits an estimated 6% of wildtype activity, indicating that this residue likely plays an important role in catalysis (11), although this 16-fold reduction in activity is much less than the 10 5 -fold reduction observed upon analysis of nucleophile mutants in mechanistically similar ␤-glycosidases (12-14), leaving some doubt concerning the role of Asp-518. This activity was not, however, based on rigorously purified enzyme, and the assay method used in this study could not distinguish this level of activity from background. Furthermore, lysosomal ␣-glucosidase and sucrase-isomaltase (Family 31) and the ␣-amylases ...

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“…Mass Spectrometry-Purified protein fractions were run on SDS-PAGE, transferred to Immobilon, digested with trypsin, and analyzed by mass spectrometry as described by Hess et al (25).…”

Section: Methodsmentioning

confidence: 99%

Identification of a Bacterial Inhibitor of Protein Kinases

Berger

Rowan

Morrison

et al. 1996

Journal of Biological Chemistry

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We show that Escherichia coli produce a factor that inhibits the activity of tyrosine and serine/threonine protein kinases. The factor is a protein found in the periplasmic compartment and is also secreted into the culture medium. Using a particle concentration fluorescence immunoassay specific for tyrosine kinase activity and inhibition of the tyrosine kinase p56 lck , we purified this factor to apparent homogeneity. Analysis of trypsindigested fragments by mass spectrometry identified the inhibitor as the bacterial periplasmic protein UDPsugar hydrolase, an enzyme with potent and nonspecific 5-nucleotidase activity. Overexpression of the enzyme in bacteria leads to coordinate increases in both 5-nucleotidase and p56 lck inhibitory activity, confirming the identity of the inhibitor. The kinase inhibitory activity appears to be due to the formation of adenosine, which we show is inhibitory for p56 lck , cAMP-dependent protein kinase, and casein kinase. Overexpression of UDPsugar hydrolase leads to an increase in the recovery of enteropathogenic E. coli following infection of HeLa cell monolayers and corresponding alterations in tyrosinephosphorylated host proteins. These results suggest that UDP-sugar hydrolase may be an important factor affecting host cell function following intracellular bacterial infection.

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Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis

Cited by 92 publications

References 32 publications

Identification and structural influence of a differentially modified N‐terminal methionine in human S l00b

Identification and structural influence of a differentially modified N‐terminal methionine in human S l00b

Unequivocal Identification of Asp-214 as the Catalytic Nucleophile of Saccharomyces cerevisiae α-Glucosidase Using 5-Fluoro Glycosyl Fluorides

Identification of a Bacterial Inhibitor of Protein Kinases

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