Methods for on-chip protein analysis |
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Authors: | Caputo Emilia Moharram Ramy Martin Brian M |
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Institution: | Unit on Molecular Structures, LNT, NIMH, NIH, DHHS, 10 Center Drive, Bldg. 10 3N309, Bethesda, MD 20892-1262, USA. |
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Abstract: | The unambiguous identification of peptides/proteins is crucial for the definition of the proteome. Using ProteinChip Array technology also known as surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS), we developed experimental protocols and probed test conditions required for the protein identification on ProteinChip surfaces. We were able to directly digest peptides/proteins on-chip surfaces by specific proteases, such as trypsin, and to obtain the peptide mass fingerprint of the sample under investigation by its direct analysis on a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry was performed on several of the resulting tryptic peptides by using collision quadrupole time of flight (Qq-TOF) MS/MS via the ProteinChip interface, thus allowing the unambiguous identification of the protein(s) within the sample. In addition, we were able to identify the C-terminal sequence of peptides by their digestion with carboxypeptidase Y directly on ProteinChip surfaces coupled with SELDI-TOF MS analysis of the resulting peptide mass ladders employing the instrument's protein ladder sequence software. Moreover, the removal of up to nine amino acid residues from the C-terminal end of a peptide extends the functional range of Qq-TOF MS/MS sequence determination to over 3000 m/z. The utility of these procedures for the proteome exploration are discussed. |
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Keywords: | Enzymatic hydrolysis ProteinChip array Surface-enhanced laser desorption/ionization C-terminal sequencing Carboxypeptidase Y |
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