Mass spectrometric study of the effects of hydrophobic surface chemistry and morphology on the digestion of surface-bound proteins.
Our previous work has demonstrated that reversed-phase chromatographic micro-beads can be used to capture proteins from complex biological matrices and the surface-bound proteins can be enzymatically digested for protein identification by mass spectrometry (MS). Here we examine the peptides generated from digestion of proteins bound to various types of micro-bead surfaces in order to determine the effects of surface chemistry and surface morphology on the digestion process. Detailed examinations of site cleavages and sequence coverage are carried out for a tryptic digestion of cytochrome c adsorbed on reversed-phase polystyrene divinylbenzene (Poros R2 beads) versus C(18) bonded-phase silica beads. It is shown that although the surface does not completely hinder the digestion of cleavage sites of the protein, the digestion products are clearly different than those obtained from a solution digest. Specifically, a partial digestion results from surface digestion, resulting in a greater number of missed cleavages than a comparable solution digest. Subsequent comparisons of peptide mass maps generated from the digestion of various proteins on surfaces with altering chemistry (C(4), C(8), C(18), and R2 beads), or with different surface morphology, were performed. The results reveal that surface chemistry plays only a minor role in affecting the peptide mass maps, and surface morphology had no noticeable effects on the resulting peptide mass maps. It is also shown that the mass spectrometric detection method used to analyze the digested peptides can significantly influence the information content on cleavage sites and the extent of sequence coverage. The use of a combination of MALDI, LC/off-line MALDI, and LC/ESI MS is demonstrated to be crucial in revealing subtle changes in the peptide mass maps.
Doucette
Alan
A
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Craft
David
D
Li
Liang
L
D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012996:Solutions / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays
D003574:Cytochrome c Group; D008565:Membrane Proteins; D010446:Peptide Fragments; D012996:Solutions; D014357:Trypsin