Besides identification also quantification of proteins can be performed by means of mass spectrometry either in data-dependent acquisition or data-independent acquisition: SWATH on the TripleTOF5600 (Sciex) and/or io nmobility-assisted data independent acquisition MS (HDMSE) on the SynaptG2Si (Waters).
For quantification by means of DDA, it is crucial to have 8-10 datapoints in the elutionprofile of a peptide. This can be obtained by adapting the cycle time.
In SWATH, the entire m/z range is divided in different ‘swaths’ or windows. The entire range of m/z values is covered in a few seconds. In each window all precursors are fragmented leading to quantitative information on all precursors. Identification of these precursors is obtained via matching of the spectra with a library built from DDA runs.
Ion mobility-assisted data independent acquisition MS or HDMSE is a second way to perform DIA experiments. In HDMSE, no windows are present but all precursors from the entire m/z range are fragmented at a certain time point. No DDA library is necessary for identification since fragments and precursors can be linked to one another through ion mobility separation. This DIA method is preferred when low amount of sample is available.
Besides label-free, quantification by means of iTRAQ or SILAC can be achieved as well.
Advantages of label-based protein quantification
– low technical variability since multiple conditions are pooled in 1 LC-MS/MS run
Advantages of label-free protein quantification
– low cost
– technical variability is minimized by the inclusion of quality controls, alignment and normalization.
Workflow
You provide us with a protein sample, free from salts, preferable with a known amount. The proteins will first be digested into peptides by means of an enzyme. For quantification purposes, internal standards can be added to the sample. Peptides are first separated by means of hydrophobicity on a C18 column and subsequently analysed by a mass spectrometer (either TripleTOF 5600, Sciex or Synapt G2Si, Waters). The spectra will be searched against a database of choice and quantitative data-analysis of PRM and HDMSE data will be performed my means of Progenesis QI-P. Swath data will be analysed by means of Peakview, Markerview and/or Skyline.