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

✍ Scribed by Zhi-Heng Huang; Tunli Shen; Jiang Wu; Douglas A. Gage; J.Throck Watson

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 162 KB
• Volume: 268
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1998.3085

✦ Synopsis

We have recently reported a simple procedure by which low picomole quantities of peptides can be modified to the corresponding N-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 g 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 acid N-hydroxysuccinimide ester (TMPP-AcOSu bromide, 2-4 nmol reagent/g protein, rt, 20 min) to provide N-terminally derivatized products, while the ⑀-NH 2 groups 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 pro-cedure 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.