Protein Sequencing by Matrix-Assisted Laser Desorption Ionization–Postsource Decay–Mass Spectrometry Analysis of theN-Tris(2,4,6-trimethoxyphenyl)phosphine-Acetylated Tryptic Digests

Abstract

We have recently reported a simple procedure by which low picomole quantities of peptides can be modified to the correspondingN-Tris(2,4,6-trimethoxyphenyl)phosphonium-acetyl (TMPP-Ac) derivatives (Z. H Huang, J. Wu, D. A. Gage, and J. T. Watson,Anal. Chem.69, 137–144, 1997). This modification significantly facilitates sequence interpretation by providing exclusively N-terminal product ions (mainly a-type ions) in the fast-atom bombardment–MS/MS and matrix-assisted laser desorption ionization–postsource decay(MALDI–PSD)–MS spectra. The TMPP-Ac derivatization approach has been extended now for the direct derivatization of tryptic digests originating from 1–5 μg of proteins with molecular weights from 10-120 kDa. Our new procedure involves tryptic digestion in aqueous solution buffered to pH 8–8.2 with phosphate or Tris–HCl, followed by reaction with TMPP-acetic acidN-hydroxysuccinimide ester (TMPP-AcOSu bromide, 2–4 nmol reagent/μg protein, rt, 20 min) to provide N-terminally derivatized products, while the ϵ-NH2groups in lysine remain unchanged. The resultant derivatized peptide mixture or its partially separated HPLC fractions are subsequently analyzed by MALDI–PSD–MS using 0.5- to 1-pmol aliquots, giving rise to product ion spectra that are easily interpretable. As there is no need for material transfer and change of buffer media, the tandem enzymatic–chemical reaction/MS analysis process is usually carried out with very high throughput (digestion, 1 h; reaction, 1/3 h; HPLC, 1 h; MALDI–PSD, 3–4 fragments/h). This procedure will be of potential use for obtaining sequence information directly from mixtures or as an adjunct of peptide mass mapping to provide protein identification with high confidence.