Protein sequencing by tandem mass spectrometry.

Abstract

Methodology for determining amino acid sequences of proteins by tandem mass spectrometry is described. The approach involves enzymatic and/or chemical degradation of the protein to a collection of peptides which are then fractionated by high-performance liquid chromatography. Each fraction, containing as many as 10-15 peptides, is then analyzed directly, without further purification, by a combination of liquid secondary-ion/collision-activated dissociation mass spectrometry on a multianalyzer instrument. Interpretation of collision-activated dissociation mass spectra is described, and results are presented from a study of soluble peptides produced by treatment of apolipoprotein B with cyanogen bromide and trypsin.Current strategy for sequencing proteins in our laboratory by tandem mass spectrometry (1) involves digestion of the protein with site-specific reagents such as cyanogen bromide or trypsin followed by fractionation of the resulting mixture by high-performance liquid chromatography (HPLC). Peptides in each fraction are then ionized by liquid secondary-ion mass spectrometry (LSIMS) (2) on a triple-quadrupole mass spectrometer. Sample is dissolved in a viscous matrix such as glycerol or monothioglycerol and then exposed to a beam of 6-to 8-keV (1 eV = 1.602 x 10-19 J) Cs' ions in the ion source of the mass spectrometer. Peptides are sputtered into the gas phase under these conditions, and the resulting mass spectrum consists largely of (M+H)+ ions characteristic of the molecular weight of each peptide in the sample (3). In a second experiment, the first quadrupole of the instrument is used to select a single (M+H)+ ion from the mixture and to transmit it to quadrupole 2, a collision chamber, where the peptide undergoes collisions with argon atoms and suffers fragmentation primarily at the various amide linkages in the molecule. The resulting fragment ions are then transferred to the third quadrupole, which separates them according to mass. The end result is a mass spectrum containing ions characteristic of the sequence of amino acids in the selected peptide. Repetition of this process under computer control provides sequence information on each peptide in the mixture. Presently, the above approach is limited by the 1800-Da mass range of our triple-quadrupole instrument. Maximum sequence information is obtained only when the protein under investigation is cleaved into peptides of molecular mass under this ceiling. New instrument developments should remedy this situation shortly (4).The major strength of the tandem mass spectrometry method is that it provides extensive sequence information over the whole length of a protein chain in a single series of experiments that involve minimal effort directed toward separation and purification of oligopeptide fragments. Total time for biochemical manipulation, HPLC fractionation, and instrumental analysis of samples from a single protein digest seldom exceeds 4 or 5 days. Even with the limited mass range of our existing instrumentation, it is usually possible ...

Bifurcating fragmentation behavior of gas-phase tryptic peptide dications in collisional activation

“…Proline is in general known to induce very strong cleavage at its N-terminal site [20], whereas the cleavage at the C-terminal site is suppressed [21]. Smith et al [17] found that in the cationic peptide VPDPR cleavage C-terminal to the proline in the second position was unusually abundant and attributed this fact to cyclization of the first two residues into a diketopiperazine structure.…”

SO2−· electron transfer ion/ion reactions with disulfide linked polypeptide ions

“…Most tandem mass spectrometers currently applied to peptide cations employ multiple relatively low energy collisions as the means for parent ion excitation [11]. Under these conditions, CID induces amide backbone cleavage, producing b-and y-type ions [12,13], which are useful in polypeptide characterization [14]. A complication of using low energy CID to study polypeptide ions generated from ESI arises when disulfide bonds are present.…”

Fragmentation of protonated ions of peptides containing cysteine, cysteine sulfinic acid, and cysteine sulfonic acid

“…Mass spectrometry has been widely used in the structure elucidation of biomolecules including peptides [13][14][15]. The effect of an acidic amino acid, i.e., aspartic acid, glutamic acid, and Cys-SO 3 H, on the fragmentation of protonated peptide ions has been well studied [16 -22].…”