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Determination of disulfide bond patterns in laminin β 1 chain N‐terminal domains by nano‐high‐performance liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight mass spectrometry
Author(s) -
Kalkhof Stefan,
Haehn Sebastian,
Ihling Christian,
Paulsson Mats,
Smyth Neil,
Sinz Andrea
Publication year - 2008
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3576
Subject(s) - chemistry , laminin , mass spectrometry , time of flight mass spectrometry , sample preparation in mass spectrometry , matrix assisted laser desorption/ionization , chromatography , mass spectrum , electrospray ionization , desorption , biochemistry , ionization , ion , extracellular matrix , organic chemistry , adsorption
The disulfide bonding patterns in the N‐terminal (LN) domains of the basement membrane protein laminin β 1 have not been investigated so far. We report an in‐depth mass spectrometric analysis using offline nano‐high‐performance liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight mass spectrometry (nano‐HPLC/MALDI‐TOF/TOF‐MS) for determining the disulfide bond patterns in the LN‐domain of recombinant mouse laminin β 1 chain for the first time. Mass spectra were recorded and the putatively disulfide‐linked peptides were subjected to LIFT‐TOF/TOF‐MS to confirm the disulfide bond. Screening the fragment ion mass spectra of disulfide‐linked peptides for characteristic 66‐amu patterns (34 u +32 u), arising from symmetric and asymmetric cleavage of disulfide bonds, facilitated their identification. Using various enzymes for proteolytic digestion of a recombinant laminin β 1 chain N‐terminal protein fragment, a linear bonding pattern of the eight cysteine residues in the LN‐domain of the laminin β 1 chain was observed with a (1‐2, 3‐4, 5‐6, 7‐8) connectivity of cysteines. The identical disulfide‐bonding pattern was found in E4, the N‐terminal laminin β 1 chain fragment derived by elastase digestion of mouse tumor laminin‐111, confirming that this pattern also occurs in native laminin. Copyright © 2008 John Wiley & Sons, Ltd.