FastCAT workflow for absolute quantification of proteins.
Target
proteins (Protein1 to Protein10) are quantified using multiple (here,
two) unpurified metabolically labeled chimeric protein standards CP1
and CP2. As an example, here CP1 contains two Q-peptides for each
of the five proteins P1 to P5; CP2 – for the proteins P6 to
P10, together with four R-peptides having native or scrambled sequences
from BSA. The amount of spiked CP1 and CP2 is adjusted to the expected
range of target protein concentrations; the concentration of BSA is
known. Proteins are digested with trypsin and analyzed by PRM LC-MS/MS.
First, CP1 and CP2 are quantified by comparing peaks of their “heavy”
reference peptides (R1CP1 to R4CP1; R1CP2 to R4CP2) and matching (R1BSA to R4BSA) peptides from BSA. Then, the concentration of each target protein
(e.g., P1) is calculated from peak areas of endogenous Q-peptides
(Q1P1 and Q2P1) and of matching “heavy”
Q-peptides (Q1CP1 and Q2CP1) whose concentration
equals CP1. Other proteins, including those covered by CP2, are quantified
similarly..