ORGANIC MASS SPECTROMETRY, VOL. 27, zyxwvutsrq 746-749 (1992) zyxwvuts In zyxwv Situ N-Phosphorylation of Oligopeptides for Fast Atom Bombardment Mass Spectrometry Hou-Jun Yang Mei-Yu He and Yun-Hua Ye Yu-Fen Zhao* Department zyxwvutsrqp of Chemistry, Tsinghua University, Beijing zyxwvuts 100084. China Department of Chemistry, Tsinghua University, Beijing 100084, China Department of Chemistry, Peking University, Beijing 100871, China Positive ion fast atom bombardment mass spectrometry (FABMS) of in situ N-phosphorylated oligopeptides showed intense quasi-molecular ions together with the successive alkene loss fragment ions, which afford multiple checks of the unequivocal reality of the relative molecular mass of the tested samples More interesting, in a novel cleavage pattern only tbe N-pbosphoryl fragment ioas gave intense peaks, the C-terminal series ions being sup pressed. For each of the N-terminal ions, losses of alkenes also occur to provide multiple cbecks for the existence of tbese ions. The FABMS of the in situ N-phospborylated oligopeptides might provide an easily accessible routine method for peptide sequencing. INTRODUCTION Fast atom bombardment mass spectrometry (FABMS), which requires only small amounts of sample, is one of the general methods for peptide sequencing. It was pointed out in a review' that in its current state FABMS of peptides suffers from several problems. First, the mass spectra of peptides usually exhibit weak or no quasi-molecular ions. Second, the N-terminal fragment ions cannot be distinguished from the C-terminal frag- ment ions in a general mass spectrum. Previously, we had reported that the FABMS of N- dialkyloxyphosphorylamino and N- dialkyloxyphosphoryl d i p e p t i d e ~ ~ - ~ showed intense molecular ions together with the A, ions produced by C(a,)-C( 1) cleavages. Moreover, the introduction of an N-dialkyloxyphosphoryl group into an amino acid resulted in an enhancement of the FABMS sensitivity by factors of 8-30.) These novel properties might provide a new approach to the analysis of peptides. In this paper, it is reported that it is possible to sequence a peptide and to determine its relative molecular mass by direct phosphorylation in situ followed by mass spectro- metric analysis. EXPERIMENTAL The FAB mass spectra were obtained with a VG ZAB-HS double-focusing mass spectrometer coupled to a PDP 11/250 computer. The neutral atom gun was operated with argon (99.995% purity) at an accelerating voltage of 8 keV, resulting in a 1 mA ion current. The pressure in the analyser was about Torr (1 0030-493X/92/060746-04 $07.00 zyxwvuts @) 1992 by John Wiley & Sons, Ltd. Torr = 133.3 Pa). Samples were applied to the matrix on a stainless-steel probe and subsequently bombarded by the neutral argon beam. Usually glycerol was used as the matrix. Immediately after the ion current had sta- bilized, ten successive scans were recorded and aver- aged, and each sample was run three times, the relative standard deviation being less than 10%. A resolving power of 3000 was maintained. Samples HGluLeuOH (la) was synthesized in our laboratory. HGluCysGlyOH (2a) was obtained from Shanghai Institute of Biochemistry, Chinese Academy of Science. CbzAlaGlyGlyOH (3d) and CbzAsp(0Bu') AlaGlyGlyGlu(OBu'), (4d) were also synthesized in our laboratory.' All the samples were characterized by FABMS and used without further purification. The N- terminal carbobenzyloxy (Cbz) groups of 3d and 46 were removed by Pb-C-catalysed hydrogenation in anhydrous methanol to give 3a and 4a, respectively. Methods In general, 3 mg of each peptide la-4a were dissolved in 0.3 ml of ethanol and 0.1 ml of water, then cooled at 0°C for 5 min. To the solution, 2.5 mol equiv. of tri- ethylamine or NaOH and 2.5 mol equiv. of carbon tetrachloride containing 1 mol equiv. of dialkyl phos- phite were added by syringe. The above mixture was stirred at 0°C for 2 h, then submitted to mass spectro- metric analysis. Each time, a sample size of 1.5 p1 was applied to the matrix used. For FABMS la4a were dissolved in ethanol at the concentration of 8 nmol pl-', and each time 1. 5 p1 of the sample solution was Received 18 July 1991 Revised manuscript received 9 December 1991 Accepted 5 February 1992