Peptide sequencing using a patchwork approach and surface-induced dissociation in sector-TOF and dual quadrupole mass spectrometers.
Surface-induced ion activation in combination with a database search strategy based on the Patchwork concept is applied to the determination of peptide sequences. Surface-induced dissociation (SID) is performed in a tandem quadrupole mass spectrometer and in a hybrid sector/time-of-flight mass spectrometer in order to evaluate the importance of accurate mass analysis of the SID fragment ions for peptide identification. The modified Patchwork approach is based on piecing together the peptide blocks in a bidirectional way, simultaneously using low-mass fragments originating from the C-terminus and N-terminus of the molecule, and relying on the measurement of the peptide's molecular weight with moderate mass accuracy. The results from this analysis are used as search filters in MASCOT's (http://www.matrixscience.com) Sequence Query search engine, with the simultaneous addition of the full MS/MS peak list. SID is performed with collision targets coated with pure and mixed composition self-assembled monolayers produced by fluorocarbon and hydrocarbon alkanethiolate solutions of varying chemical composition. The resulting MS/MS spectra produced on pure and mixed hydrocarbon SAMs are submitted to the modified version of Patchwork sequencing. It is found that hydrocarbon surfaces improve the relative abundance of larger fragments. Under the moderate mass accuracy conditions (+/-0.3 u) offered by our linear-TOF-SID instrument, it is found that increasing the abundance of larger fragments dramatically improves the sequencing scores.
Fernández
Facundo M
FM
Department of Chemistry, University of Arizona, Tucson, Arizona 85721-0041, USA.
Smith
Lori L
LL
Kuppannan
Krishnamoorthy
K
Yang
Xi
X
Wysocki
Vicki H
VH
D000465:Algorithms; D030562:Databases, Protein; D005466:Fluorocarbons / Q000737:chemistry; D006838:Hydrocarbons / Q000737:chemistry; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis*; D020539:Sequence Analysis, Protein / Q000379:methods; D013499:Surface Properties
D005466:Fluorocarbons; D006838:Hydrocarbons; D007202:Indicators and Reagents; D010455:Peptides