Limited acid hydrolysis as a means of fragmenting proteins isolated upon ProteinChip (R) Array surfaces

Citation
Sh. Lin et al., Limited acid hydrolysis as a means of fragmenting proteins isolated upon ProteinChip (R) Array surfaces, EUR J MASS, 7(2), 2001, pp. 131-141
Citations number
18
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Spectroscopy /Instrumentation/Analytical Sciences
Journal title
EUROPEAN JOURNAL OF MASS SPECTROMETRY
ISSN journal
1469-0667 → ACNP
Volume
7
Issue
2
Year of publication
2001
Pages
131 - 141
Database
ISI
SICI code
1469-0667(2001)7:2<131:LAHAAM>2.0.ZU;2-9
Abstract
ProteinChip Array technology enables protein purification, protein profilin g and biomarker discovery on a convenient biochip platform. Traditional pro teomic approaches towards protein identification rely upon the generation o f peptides through the use of specific proteases. However, for a variety of reasons, the digestion of proteins bound to planar arrays using specific p roteases, such as trypsin, has proven to be difficult, at times providing l ittle or no protein digestion at all. Additionally, should more than one pr otein be present on the array surface, the digestion product consists of pe ptides from different proteins, adding another dimension of complexity to d atabase mining approaches. These factors have driven our group to explore a lternative means of on-chip protein digestion. In this article, we describe an approach to generating peptide maps by limited acid hydrolysis. Dependi ng upon the adsorbed protein, this method requires between 500 femtomol to 5 picomol of protein for on-chip hydrolysis. Besides generating several int ernal peptide fragments, limited acid hydrolysis also has the advantage of generating peptide ladders from the N- or C-terminus of the protein. From t hese ladders, a partial primary sequence of the protein can be directly der ived using analysis by a simple laser desorption/ionization mass spectromet er. Furthermore, tandem mass spectrometry (MS/MS) can be performed on sever al internal peptide fragments, thus facilitating the identification of seve ral proteins within a mixture. Based upon the preliminary results of this w ork, we continue to explore the possibility of using limited acid hydrolysi s to identify unknown proteins captured on ProteinChip Array surfaces.