Journal: The Journal of Cell Biology
Article Title: Spindle Checkpoint Protein Xmad1 Recruits Xmad2 to Unattached Kinetochores
Figure Lengend Snippet: ( A ) Xmad2 associates with an 85-kD protein in CSF-arrested extracts. Immunoprecipitation was performed by using a control antibody (lanes 1 and 3 ) or an anti-Xmad2 antibody (lanes 2 and 4 ) as indicated. The immunoprecipitates were washed with a buffer containing 1% Triton X-100 (lanes 1 and 2 ) or 0.1% SDS (lanes 3 and 4 ). The proteins were resolved by SDS-PAGE and stained with Coomassie blue. The migration of p85, IgG, and Xmad2 is indicated on the left, and that of molecular weight standards on the right. ( B ) Sequencing of Xmad1. The unseparated pool of tryptic peptides recovered from the gel matrix was analyzed using a novel quadrupole TOF mass spectrometer. ( a ) Mass spectrum of the unseparated tryptic digest of the Xmad1 band. The peptide ions designated with T were in turn isolated by a quadrupole mass analyzer, fragmented in the collision cell, and then their tandem mass spectra were acquired with a reflector time-of-flight module. Peaks designated with asterisks are trypsin autolysis products. The peptide ion designated with A belongs to the antibody used in the purification of Xmad1. ( b ) Tandem mass spectrum of the doubly charged peptide ion T 8 (marked with an arrow in a ). Upon collisional fragmentation, tryptic peptides tend to produce a continuous series of fragment ions containing the COOH terminus (Y″ ions; Roepstorff and Fohlman, 1984). The peptide sequence (shown above) is deduced by considering precise mass differences between adjacent Y″ ions. M *, N -acetylated methionine sulfoxide amino acid residue. The spectrum also contains minor series of the NH 2 -terminal fragment ions (B ions) as well as internal fragment ions. Therefore to deduce the sequence with high confidence it is necessary to distinguish Y″ ions from the other ions in the spectrum. This has been achieved by selective isotopic labeling of the COOH-terminal carboxyl group of the peptides. ( c ) A zoom of the region in the tandem mass spectrum of T 8 shows the principal of sequence readout of isotopically labeled peptides. Upon tryptic cleavage of the protein in a buffer containing H 2 16 O/H 2 18 O 1:1 (vol/vol) COOH-terminal carboxyl groups of peptide molecules incorporate 16 O and 18 O atoms in a 1:1 ratio. Thus the fragment ions containing the COOH terminus of the peptide (mostly Y″ ions) appear in the fragment spectrum as a characteristic isotopic pattern—a doublet split by 2 D. B ions or other fragment ions not containing COOH-terminal carboxyl group are observed as ions having normal isotopic pattern.
Article Snippet: The unseparated pool of tryptic peptides recovered from the gel matrix was sequenced on a experimental prototype of a hybrid quadrupole Time-Of-Flight tandem mass spectrometer developed by PE Sciex (Ontario, Canada) and University of Manitoba (Winnipeg, Canada) ( ).
Techniques: Immunoprecipitation, SDS Page, Staining, Migration, Molecular Weight, Sequencing, Mass Spectrometry, Isolation, Purification, Isotopic Labeling, Labeling