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, PROTEOMICS, 1(9), 2001, pp. 1172-1184
Citations number
18
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Chemistry & Analysis
Journal title
PROTEOMICS
ISSN journal
1615-9853 → ACNP
Volume
1
Issue
9
Year of publication
2001
Pages
1172 - 1184
Database
ISI
SICI code
1615-9853(200109)1:9<1172:LAHAAM>2.0.ZU;2-6
Abstract
ProteinChip (R) array technology enables protein purification, protein prof iling, and biomarker discovery on a convenient biochip platform. Traditiona l proteomic approaches towards protein identification rely upon the generat ion of peptides through the use of specific proteases. However, for a varie ty of reasons, the digestion of proteins bound to planar arrays by specific proteases, such as trypsin, has proven to be difficult, at times providing little or no protein digestion at all. Additionally, should more than one protein be present on the array surface, the digestion product consists of peptides from different proteins, adding another dimension of complexity to database mining approaches. These factors have driven our group to explore alternative means of on-chip protein digestion. In this article, we descri be an approach to generate peptide maps by limited acid hydrolysis. Dependi ng upon the adsorbed protein, this method requires between 500 femtomole to 5 picomole of protein for on-chip hydrolysis. Besides generating several i nternal peptide fragments, limited acid hydrolysis also has the advantage o f generating peptide ladders from the N- or C-terminus of the protein. From these ladders, partial primary sequence of the protein can be directly der ived when analyzed by a simple laser desorption/ionization mass spectromete r. Furthermore, tandem mass spectrometry can be performed on several intern al peptide fragments, thus facilitating the identification of several prote ins within a mixture. Based upon the preliminary results of this work, we c ontinue to explore the possibility of using limited acid hydrolysis to iden tify unknown proteins captured on ProteinChip array surfaces.