Mapping and sequence‐specific identification of phosphopeptides in unfractionated protein digest mixtures by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

FEBS Letters

Hutchens, T.William¹  Yip, Tai-Tung¹ 

[1] Protein Structure Laboratory, USDA/ARS Children's Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates, Houston, TX 77030, USA

关键词: Protein sequence;    Phosphorylation;    Phosphopeptide;    Casein;    Mass spectrometry;    Laser desorption;   

DOI  :  10.1016/0014-5793(92)81264-M

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We have demonstrated a procedure for the rapid (minutes), sensitive (sequence-specific identification of phosphopeptides in unfractionated digests of phosphoproteins using matrix-assisted UV laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. The mass-dependent identification of one specific 13-residue phosphopeptide (S105-K117), observed among the 153 possible trypsin digest fragments of human β-casein (211 residues), was confirmed by amino acid sequence analysis of the ^???P-labeled peptide after isolation by reverse-phase HPLC. MALDI-TOF was also used to monitor the rate and extent to which an 18-residue N-terminal β-casein peptide (R1-K18) was phosphorylated in vitro. These results demonstrate that MALDI-TOF may be used (i) to facilitate the identification of sequence-specific sites of protein phosphorylation and dephosphorylation. (ii) to monitor protein and peptide phosphorylation and dephosphorylation reaction rate, even in complex unfractionated mixtures, (iii) to determine the minimum primary structure necessary for the phosphorylation of specific protein surface domains, and (iv) to evaluate the effects of intact protein phosphorylation and dephosphorylation on susceptibility to subsequent proteolytic events.

【 授权许可】

Unknown