Biomedical Engineering Reference
In-Depth Information
Protein Identification
"shotgun digest"
MS/MS
peptide fragmentation
observed pattern
Proteins
Peptides
HPLC
MS
peptides
statistical pattern
match
theoretical (database) pattern
b2
y2
b3
y3
b4
y4
b5
y5
b6
y6
Figure 13.2
Schematic of a typical LC/MS/MS 'shotgun' experiment. Proteins are digested
into peptides using a site-specific protease and the resulting peptides are resolved by HPLC
directly in line with tandem MS. Individual fragmentation patterns from tandem MS spectra are
then searched against theoretical patterns generated by database search algorithms to produce
statistically significant candidate protein identifications.
cysteine-containing peptide enrichment step using a biotin-streptavidin interaction). With
this approach, quantification is performed on the basis of the ion intensity of the intact
peptides, which can be obscured by co-eluting peptides and affected by low signal noise.
Another quantitative strategy involves the use of similar stable isotope tags that contain
isobaric reporter ions that are indistinguishable in the mass spectrometer until a selected
peptide is fragmented during tandem MS. Termed isobaric tag for relative and absolute
quantitation (iTRAQ) [34] or tandem mass tags [35], differentially labeled samples can be
co-resolved in a single LC/MS/MS analysis, and the relative quantification can be derived
from the ion intensities of the reporter fragmentation ions (since they are indistinguishable
in the intact peptide, they are all selected for simultaneous fragmentation during MS/MS).
More recently, the LC/MS/MS field has seen an increase in the use of so-called 'label-free'
approaches, where individual samples are analyzed by LC/MS/MS separately, and relative
protein abundance is related between samples using the peptide MS/MS fragmentation spec-
tra that give rise to statistically significant protein identifications (termed 'spectral counting')
[36, 37]. Whereas these approaches are suitable for global discovery-phase experiments,
selected single-reaction monitoring/multiple-reaction monitoring (SRM/MRM) is another
label-free LC/MS/MS technique, whereby a collection of previously selected peptides can
be targeted for subsequent (or validation) quantitative analyses [38, 39].
13.2.2.1 Multidimensional LC/MS/MS strategies: Procedure and applications
•
Resolution of peptides derived from proteolytic cleavage.
•
Based on peptide-fragmentation patterns from MS data followed by database searching.
•
Involves RP-HPLC separations directly in-line with tandem MS of peptides derived from
proteins via controlled proteolytic cleavage.
