Median-Based Absolute Quantification of Proteins Using Fully Unlabeled Generic Internal Standard (FUGIS)

Abstract

By reporting the molar abundance of proteins, absolute quantification determines their stoichiometry in complexes, pathways, or networks. Typically, absolute quantification relies either on protein-specific isotopically labeled peptide standards or on a semiempirical calibration against the average abundance of peptides chosen from arbitrarily selected proteins. In contrast, a generic protein standard FUGIS (fully unlabeled generic internal standard) requires no isotopic labeling, chemical synthesis, or external calibration and is applicable to quantifying proteins of any organismal origin. The median intensity of the peptide peaks produced by the tryptic digestion of FUGIS is used as a single-point calibrant to determine the molar abundance of any codigested protein. Powered by FUGIS, median-based absolute quantification (MBAQ) outperformed other methods of untargeted proteome-wide absolute quantification.

FastCAT Accelerates Absolute Quantification of Proteins Using Multiple Short Nonpurified Chimeric Standards

“…However, the abundance of different CPs should be referenced to the same spiked-in BSA standard. We achieved it by including reference peptides with the scrambled sequences (R s -peptides) 33 that, nevertheless, elicit a very similar response in MS1 spectra compared to the corresponding native R n -peptides. For better sensitivity, the targeted analysis would also require PRM for the quantification of CPs in the same LC-MS/MS run.…”

FastCAT accelerates absolute quantification of proteins by using multiple short non-purified chimeric standards

“…However, the abundance of different CPs should be referenced to the same spiked-in BSA standard. We achieved it by including reference peptides with the scrambled sequences (R s -peptides) 33 that, nevertheless, elicit very similar response in MS1 spectra compared to corresponding native R n -peptides. However, we reasoned that, for better sensitivity, the targeted analysis would require PRM also for quantifying CPs in the same LC-MS/MS run.…”

A framework for quality control in quantitative proteomics

“…It is important to note that the use of internal and external QCs should be separate from signal calibration. The use of a reference material for single point calibration has been described by our group and others [85][86][87] . We have used internal QCs and the external inter-batch and inter-experiment QCs to evaluate the performance of normalization, signal calibration, or batch correction.…”