Abstract
Proteomics has opened a new horizon in biological sciences. Global proteomic analysis is a promising technology for the discovery of thousands of proteins, post-translational modifications, polymorphisms, and molecular interactions in a variety of biological systems. The activities and roles of the identified proteins must also be elucidated, but this is complicated by the inability of conventional proteomic methods to yield quantitative information for protein expression. Thus, a variety of biological systems remain “black boxes”. Quantitative targeted absolute proteomics (QTAP) enables the determination of absolute expression levels (mol) of any target protein, including low-abundance functional proteins, such as transporters and receptors. Therefore, QTAP will be useful for understanding the activities and roles of individual proteins and their differences, including normal/disease, human/animal, or in vitro/in vivo. Here, we describe the study protocols and precautions for QTAP experiments including in silico target peptide selection, determination of peptide concentration by amino acid analysis, setup of selected/multiple reaction monitoring (SRM/MRM) analysis in liquid chromatography–tandem mass spectrometry, preparation of protein samples (brain capillaries and plasma membrane fractions) followed by the preparation of peptide samples, simultaneous absolute quantification of target proteins by SRM/MRM analysis, data analysis, and troubleshooting. An application of QTAP in biological sciences was introduced that utilizes data from inter-strain differences in the protein expression levels of transporters, receptors, tight junction proteins and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice. Among 18 molecules, 13 (abcb1a/mdr1a/P-gp, abcc4/mrp4, abcg2/bcrp, slc2a1/glut1, slc7a5/lat1, slc16a1/mct1, slc22a8/oat3, insr, lrp1, tfr1, claudin-5, Na+/K+-ATPase, and γ-gtp) were detected in the isolated brain capillaries, and their protein expression levels were within a range of 0.637-101 fmol/μg protein. The largest difference in the levels between the three strains was 2.2-fold for 13 molecules, although bcrp and mct1 displayed statistically significant differences between C57BL/6J and the other strain(s). Highly sensitive simultaneous absolute quantification achieved by QTAP will increase the usefulness of proteomics in biological sciences and is expected to advance the new research field of pharmacoproteomics (PPx).
Highlights
Proteomics by name is almost 20 years old and has rapidly grown into one of the most active research areas in biological sciences
We have previously established an liquid chromatography (LC)-Mass spectrometry (MS)/MS quantification method for more than 500 proteins including human, monkey, and mouse transporters, enzymes, and receptors, and we have reported quantitative protein expression profiles in brain capillaries, livers, kidneys, platelets, plasma, meningioma, human blood–brain barrier (BBB) model cell lines, human umbilical vein endothelial cell lines (HUVECs), human pancreatic adenocarcinoma cell lines, and human breast and stomach cancer cell lines [4,5,6,7,11,12,13,14,15,16,17,18]
Setup of LC-MS/MS quantification system for Selected/multiple reaction monitoring (SRM/MRM) analysis Here, we describe the principle of SRM/MRM analysis and how to optimize the analytical conditions for peptide quantification in the SRM/MRM mode of HPLC-Triple quadrupole mass spectrometer (QqQ), which is one of the most routinely used configurations of LC and MS for quantification
Summary
Proteomics by name is almost 20 years old and has rapidly grown into one of the most active research areas in biological sciences. Mass spectrometry (MS)-based protein identification is widely adopted, and recent advances in MS and global proteomics (Figure 1), including protein sequence databases, have enabled the identification of hundreds to thousands of proteins in biological materials in a single analysis [1,2,3]. Abundant proteins are easy to identify, but low-abundance proteins are difficult to detect due to high background noise when analyzing complex samples (Figure 1). Relevant molecules with low protein expression levels, such as transporters and receptors, are not readily identified by current global proteomic technologies. The activities and roles of individual proteins must be elucidated, but this is hampered by the lack of quantitative information for protein expression in global proteomics. Quantitative targeted absolute proteomics (QTAP) represents a new generation of proteomic methods that
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