A universal SI-traceable isotope dilution mass spectrometry method for protein quantitation in a matrix by tandem mass tag technology

Abstract

Isotope dilution mass spectrometry (IDMS), an important metrological method, is widely used for absolute quantification of peptides and proteins. IDMS employs an isotope-labeled peptide or protein as an internal standard although the use of a protein provides improved accuracy. Generally, the isotope-labeled protein is obtained by stable isotope labeling by amino acids in cell culture (SILAC) technology. However, SILAC is expensive, laborious, and time-consuming. To overcome these drawbacks, a novel universal SI-traceable IDMS method for absolute quantification of proteins in a matrix is described with human transferrin (hTRF). The hTRF and a human serum sample were labeled with different tandem mass tags (TMTs). After mixing the TMT-labeled hTRF and serum sample together followed by digestion, the peptides were separated by nano-liquid chromatography and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Using the signature peptides, we calculated the ratios of reporter ions from the TMT-labeled peptides which, in turn, allowed determination of the mass fraction of hTRF. The recovery ranged from 97% to 105% with a CV of 3.9%. The LOD and LOQ were 1.71 × 10(-5) g/g and 5.69 × 10(-5) g/g of hTRF in human serum, respectively, and the relative expanded uncertainty was 4.7% with a mass fraction of 2.08 mg/g. For comparison, an enzyme-linked immunosorbent assay (ELISA) method for hTRF yielded a mass fraction of 2.03 mg/g. This method provides a starting point for establishing IDMS technology to accurately determine the mass fractions of protein biomarkers in a matrix with traceability to SI units. This technology should support the development of a metrological method useful for quantification of a wide variety of proteins.

Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

“…Most of them rely on metabolic labeling and purification of a recombinant version of the protein of interest, or a shorter specific protein sequence, like the Protein Standards for Absolute Quantification (PSAQ) (21), absolute SILAC (22), PrEST (23), FlexiQuant (24), and TAQSI (25) methods. Another approach consisting in the chemical labeling of proteins using isobaric stable isotope tagging technology (26) has recently been described to reduce costs and the time required to grow cells in heavy amino acid-containing media. In all these approaches, because the internal standard is processed together with its endogenous analog throughout the whole workflow, accuracy is markedly better than achievable with AQUA and QconCAT approaches when determining absolute quantities of proteins in various matrices (17).…”

DNA Adducts as Biomarkers To Predict, Prevent, and Diagnose Disease—Application of Analytical Chemistry to Clinical Investigations

“…103−107 Formaldehyde covalently modifies dA, forming N 6 -hydroxymethyl-dA (N 6 -HOMe-dA, 13) and dG forming N 2 -hydroxymethyl-dG (N 2 -HOMe-dG, 14). 108−111 Treatment of samples with NaCNBH 3 during DNA hydrolysis reduces the Schiff's base to form stable versions of these adducts, N 6 -Me-dA (15) and N 2 -Me-dG (16), which can be measured using MS. 112 Analysis of adducts in cells exposed to formaldehyde revealed N 2 -HOMe-dG to be the primary DNA adduct formed, with no detectable formation of N 6 -HOMe-dA. 111 N 6 -Me-dA has been measured in leukocyte DNA from smokers and nonsmokers using LC−MS/MS (QQQ; LOD: 0.3 fmol).…”

“…With advances in mass spectrometry, it is much more quantitative and precise to measure adducts using analytical methods. In addition, isotopic standards provide the highest metrological standards for measuring biomolecules . As described for 8-oxo-dG, highly sensitive, specific, reproducible, and robust methods are essential for establishing DNA adducts as biomarkers in clinical testing.…”

“…In addition, isotopic standards provide the highest metrological standards for measuring biomolecules. 16 As described for 8-oxo-dG, highly sensitive, specific, reproducible, and robust methods are essential for establishing DNA adducts as biomarkers in clinical testing. Within the past decade, MS methods have been developed that meet these requirements and have been used to define the relationship between adduct levels and disease.…”

Quantitative Proteomics in Translational Absorption, Distribution, Metabolism, and Excretion and Precision Medicine

“…A known and consistent amount of SIL peptides is spiked into all samples, and the ratio is monitored to estimate the absolute levels. However, isotope dilution assay can only be used for a limited number of proteins because of the cost (Li et al, 2016;Vildhede et al, 2018;Wi sniewski et al, 2019). AQUA, QconCAT, and PSAQ all use the isotope dilution or spiked-in standards, where AQUA standards consist of synthetic peptides that are spiked into the sample after proteolysis (Gerber et al, 2003), QconCAT concatemers are chimeric proteins that are composed of different proteotypic peptides from multiple target proteins (Beynon et al, 2005), and PSAQ standards are full-length protein(s) that match the biochemical properties of the target protein(s) and spiked in at the beginning of the analytical process (Kaiser et al, 2011).…”