Analysis of Post-translational Modifications by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 95

using one or more of a number of available fragmentation mecha-
nisms; (ii) removal of the modification between consecutive mass
spectrometric analyses; (iii) selective enrichment of modified pro-
teins or peptides based on the modified functional group prior to
MS/MS, and (iv) PTM-specific multistage MS strategies. Tan-
dem MS (MS/MS) as used in a typical product ion analysis
( Fig. 5.1 ) refers to the mass spectrometric analysis of all ions
in a sample, prior to the subsequent selection and fragmenta-
tion of an ion of a specific m / z, and the subsequent MS anal-
ysis of the resultant fragment ions. When applied to peptides,
both the amino acid sequence and the type and site of mod-
ification can be determined. Large-scale characterization of the
proteins present in a complex sample, such as a cell lysate, is typi-
cally performed using a standard “bottom-up” proteomics work-
flow, where proteins are digested using a suitable protease (most
commonly trypsin) generating smaller analytes better suited to
current MS analysis workflows. These peptides are then fraction-
ated using one or more forms of liquid chromatography [e.g.,
strong cation exchange (SCX) and reverse phase (RP)] prior to
Fig. 5.1. PTM-targeted MS/MS strategies. ( i ) Precursor ion scanning: spectra are recorded only for those modified
peptide ion precursors (indicated by ∗ ) that generate a characteristic product ion following CID at an m/z specific for
the modification of interest; ( ii ) neutral loss-triggered MS/MS/MS: CID of a modified peptide ion precursor (indicated
by ∗ ) which results in predominant loss of a neutral mass in the product ion spectrum (indicated as ) can be used to
trigger either CID of the molecular ion minus the modification (MS/MS/MS) or MS/MS of the modified peptide ion using
ETD; ( iii ) selected/multiple reaction monitoring tandem MS analyses can be used to trigger CID of a modified peptide
ion precursor of defined m/z (indicated by ∗ ) following the detection of a positive transition of interest demonstrating
modification.