Use of nonspecific cleavage products for protein sequence analysis as shown on calcyclin isolated from human granulocytes

In this paper, analysis strategies developed for a sequencing problem concerning the identification of an S100 protein isolated from human granulocytes are discussed. The analysis of a trypsinized lyophilized sample suggested the presence of a number of peptides which are non-tryptic in origin. Duri...

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Bibliographic Details
Published in:Journal of the American Society for Mass Spectrometry 2001-11, Vol.12 (11), p.1180-1185
Main Authors: König, Simone, Zeller, Martin, Peter-Katalinic, Jasna, Roth, Johannes, Sorg, Clemens, Vogl, Thomas
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Biological activity
Cell Cycle Proteins
Cleavage
Desorption
Electrophoresis, Polyacrylamide Gel
Granulocytes
Granulocytes - chemistry
High performance liquid chromatography
Humans
Hydrolysis
In Vitro Techniques
Ionization
Ions
Mass spectrometry
Molecular Sequence Data
Peptide Fragments - chemistry
Peptide Fragments - isolation & purification
Peptide Mapping
Peptides
Proteins
S100 Calcium Binding Protein A6
S100 Proteins - chemistry
S100 Proteins - isolation & purification
Sequence Analysis - methods
Trypsin
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Summary:In this paper, analysis strategies developed for a sequencing problem concerning the identification of an S100 protein isolated from human granulocytes are discussed. The analysis of a trypsinized lyophilized sample suggested the presence of a number of peptides which are non-tryptic in origin. During purification of proteins from cell lysates nonspecific cleavage can be observed which may reflect biological processes and can become an unavoidable analytical problem. Current mass spectrometric software is evaluated for the analysis of nonspecific digests in this context. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), high-performance liquid chromatography (HPLC)-MS/MS, and selected ion monitoring (SIM)-MS/MS have been used for peptide analysis and in addition HPLC-MS was carried out for protein analysis leading to the detection of an N-terminal modification of the protein. The success of the study is mainly due to the careful investigation of nonspecific cleavage products. Data obtained from the routine mass spectrometric analysis of an in-gel-digest allowed the identification of this protein as S100 calcium-binding protein A6–calcyclin whose expression in granulocytes has not been described so far.
ISSN:1044-0305
1879-1123
DOI:10.1016/S1044-0305(01)00300-2