- Identification and quantification of proteins
- Non-VCIID investigators are generally referred to the VGN Proteomics Facility for routine proteomics measurments.
- The Mass Spectrometry Facility does offer proteomics measurements by complementary methods to the VGN facility. Differences in measurement approach are shown below:
- All measurements are made at high resolution on the Waters Xevo G2-XS QTOF, not on low resolution linear ion traps.
- Measurements are made using Waters MSE for MS/MS data independent acquisition, not data dependent acquisition.
- Absolute quantification of proteins is performed using addition of a protein standard that does not require expensive stable isotopic labeling.
- Peptide and protein identifications and quantification are performed using Protein Lynx Global Server (PLGS) and Nonlinear Progenesis QI
- PLGS provides provision of identifying unidentified peptides. Unidentified peptides can be identified by de novo sequencing tools available in PLGS. This approach is helpful for identifying chemical cross-linking of proteins.
- Separations are performed on 1-mm or 300-µm UPLC columns that have higher capacities for tolerating complex protein mixtures, such as cell lysates. This approach allows determination of femtomole amounts of minor proteins.
- Identification and quantification of postranslational modifications (PTM) of proteins
- Non-VCIID investigators are generally referred to the VGN Proteomics Facility for routine PTM measurments, such as phosphorylation.
- The Mass Spectrometry Facility does offer PTM measurements of non-routine, difficult to analyze PTMs that are not routinely accepted in the VGN facility. A primary example is analysis of glycosylation patterns. Glycosylation patterns are measured on the peptides without release of the glycans.
- The Mass Spectrometry Facility will discuss speific PTM measurements with investigators and develop methods as needed.
- Quantification of peptides and PTMs (and proteins) by TSQ and SRM targeted analyses
- The VGN Proteomics Facility does not offer SRM measurements by TSQ.
- Stable isotopically labeled peptides matching the peptide to be quantified with and without the PTM are used.
- The primary PTM quantified by this method is phosphorylation for which isotopically labeled phosphorylated peptides can be obtained.
- Proteoform measurement of whole proteins
- These measurements are complementary to PTM measurement and can be done in conjunction with peptide digest PTM analysis. The latter provides specific site information of PTMs. The proteoform measurement of the whole protein provides information about the fraction of proteins that have a particular PTM combination.
Last modified October 08 2015 02:24 PM