Quantitative Performance of Internal Standard Platforms for Absolute Protein Quantification Using Multiple Reaction Monitoring-Mass Spectrometry

Abstract

Stable-isotope-labeling mass spectrometry involves the addition of known quantities of stable-isotope labeled standards, which mimic native molecules, to biological samples. We evaluated three conventional internal standard platforms (synthetic peptides, QconCAT constructs, and recombinant proteins) for quantitative accuracy, precision, and inherent advantages and limitations. Internal standards for the absolute quantification of three human cytokine proteins (interferon gamma, interleukin-1 beta, and tumor necrosis factor alpha) were designed and verified. Multiple reaction monitoring assays, calibration curve construction, and regression analysis were used to assess quantitative performance of the internal standard platforms. We also investigated a strategy for methodological improvement to current platforms using natural flanking sequences. Data analysis revealed that full length protein standards have the broadest quantitative reliability with accuracy being peptide-dependent for QconCATs and synthetic peptides. Natural flanking sequences greatly improved the quantitative performance of both QconCAT and synthetic peptide standards.

Impact of Sample Matrix on Accuracy of Peptide Quantification: Assessment of Calibrator and Internal Standard Selection and Method Validation

“…This is not surprising as trypsin has been shown to preferentially cleave proteins at certain cleavage sites 34 and peptide specific digestion time courses have been shown before. 8,35 Yet, a need for a long trypsin digestion (>15 hours) was considered a liability as it will increase the probability of non-specific protein cleavage 36 and peptide modifications. 37,38 Therefore, the YIL peptide was excluded as a signature peptide.…”

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry–Based Assays

“…Importantly, the same potential limitation also applies when using isotopically-labeled proteins as internal standards. While potentially providing improved precision and less bias than labeled peptides in quantitative MS-based assays (8, 10), differences in the repertoire of modifications (e.g., phosphate, carbohydrate, ubiquitin, etc.) on amino acid residues near enzymatic cleavage sites, for example, can alter digestion and recovery of desired analyte peptides from internal standard proteins.…”

Ten years of QconCATs: Application of multiplexed quantification to small medically relevant proteomes

“…In animals, quantitative assays based on QconCAT were used to study variations in inflammatory protein expression in tissues, body fluids and cells [19,20]. Other studies applied the QconCAT strategy to the quantification of multiple major urinary proteins (MUPs) [21], human liver drug-metabolizing enzymes and drug transporters [15,22], human intestinal transporters [23], and human cytokine proteins [24]. Chen et al used QconCAT standards to quantify proteins, in post-mortem human brain tissue, which are related to the development of Alzheimer's disease including clusterin and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), phosphatidylinositol-binding clathrin assembly protein (PICALM) and amyloid precursor protein (APP) [25][26][27][28].…”