Peptide Ion Fragmentation Technologies CID = collision induced dissociation CAD = collision...
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Transcript of Peptide Ion Fragmentation Technologies CID = collision induced dissociation CAD = collision...
Peptide Ion Fragmentation TechnologiesCID = collision induced dissociationCAD = collision activated disassociation
HCD = high energy collisional disassociation + high resolution fragments in MS2 spectra
e.g. +/- 0.01Da MS2 window or ~15ppmsCID/CAD = low resolution fragments +/- 0.5Da
+ can be acquired much more quickly
Comparison of single-scan (500- to 600-msec) CAD and ETD mass spectra recorded during data-dependent analyses (nHPLC-μESI-MS/MS) of phosphopeptides generated in a tryptic
digest of human nuclear proteins.
Syka J E P et al. PNAS 2004;101:9528-9533
©2004 by National Academy of Sciences
ETD = Electron Transfer Disassociation
c and z* ions primarily
Can handle phosphorylation better.
A protein domain is a conserved part of a given protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded.
PFAM
Database of hidden markov models (HMMs) that can be used to find domains by sequence alone.
Predicted Domain Interaction Phosphosite
Highly conserved across species.
Selection on mutants of the site and IP experiment confirms interaction.Indicates phosphorylation promotes weaker binding.
SAM which activates downstream MET4 relieves site mutant.
Phosphorylation is Usually Conserved at an Interface but the Site Position Might Change
Can’t rule out the possibility they’re missing phosphosites due to the limited coverage of the mass spec data
SSA1 (HSP70 family protein) Highly abundant protein
A little strange they refer to HSP70 as a “domain”
Nevertheless in each region they
D mutants mimicthe phosphoyrlated state of theamino acid
Mutating Phosphosites Slows Down the Growth of Yeast Cells Measured 2 ways. Yeast cells also lack SSA2-
4 (paralogs)
Protein abundance is the same.