Peptide-Mass Profiles of Polyvinylidene Difluoride-Bound Proteins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry in the Presence of Nonionic Detergents

✍ Scribed by Farzin Gharahdaghi; Michele Kirchner; Joseph Fernandez; Sheenah M. Mische

• Publisher: Elsevier Science
• Year: 1996
• Tongue: English
• Weight: 97 KB
• Volume: 233
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1996.0012

✦ Synopsis

enzymatic digestion of the PVDF-bound protein is nec-Matrix-assisted laser desorption/ionization time-ofessary to obtain peptides for sequence analysis (4-12). flight mass spectrometry (MALDI-TOF MS), in con-Methods are available for the digestion of proteins from junction with enzymatic digestion of proteins and moeither gel or PVDF, both providing acceptable recovery lecular weight search of peptide-mass database is a of peptides (4-15). However, the major advantage of powerful technique for peptide/protein identifica-PVDF-bound protein is that it can be sequenced dition. Ideally, peptide mixtures should be compatible rectly, whereas gel samples cannot. with both MALDI-TOF and microsequencing. In our Regardless of the medium used, the ideal digestion laboratory, enzymatic digestion and extraction of method should (1) recover peptides in high yield, (2) peptides from polyvinylidene difluoride (PVDF)recover longer peptides for Edman sequence analysis bound proteins is performed in the presence of non-(important for unknown proteins because more contigionic detergents. However, nonionic detergents have uous protein sequence data allow for better DNA probe been shown to cause signal suppression in MALDIdesign for subsequent screening of libraries), and (3) TOF analysis. This study demonstrates that by using recover peptides in an extraction buffer that is compatia modified matrix solution, peptide-mass fingerprintble with other microanalytical methods (i.e., RP-HPLC, ing of PVDF-bound proteins by MALDI-TOF can be obtained in the presence of nonionic detergents such MALDI-TOF MS). Based on the published literature of as hydrogenated Triton X-100 (RTX-100), octylglucomethods used for the digestion and elution of peptides pyranoside, and Tween 20.