A practical guide to the FLEXIQuant method.
A protein molecule exists as a heterogeneous population of posttranslationally modified forms, which are of potential interest to biologists. However, due to detection or methodology limitations, they remain uncharacterized. When a protein does become a prioritized interest in a laboratory, workflows aimed for its purification and characterization are implemented. Inherent in these workflows is the enrichment of the protein from the biological lysate, rendering it an ideal sample for mass spectrometry (MS), as detection of several peptides is greatly increased. In order to capitalize on this enhanced detection of the protein of interest, we have developed a full-length expressed protein quantification standard (FLEXIQuant standard) that is in vitro synthesized, devoid of posttranslational modifications (PTMs), and implemented into the purification workflow of the endogenous counterpart-as such it serves as an internal MS standard. FLEXIQuantification allows for the unbiased identification of peptides undergoing PTM as a function of a particular biological state. The extent of PTM is also quantified, providing further insight into the regulation of the protein.
Department of Pathology, Children's Hospital Boston and Harvard Medical School, Boston, MA, USA.
Sasha
S
Singh
Marc
M
Kirchner
Judith A
JA
Steen
Hanno
H
Steen
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000592:standards*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins; D012015:Reference Standards; D014357:Trypsin / Q000737:chemistry
D010446:Peptide Fragments; D020543:Proteome; D011993:Recombinant Fusion Proteins; D014357:Trypsin