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Editorial |
Department of Laboratory Medicine, National Institutes of Health, Building 10, Room 2C-407, Bethesda, MD 20892, Fax 301-402-1885, E-mail ghortin{at}mail.cc.nih.gov.
In this issue of Clinical Chemistry, Bondar and coworkers at the Mayo Clinic report on development of a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for zinc-
2-glycoprotein (1). The assay described may be more notable for the approach employed rather than for the specific analyte. Unlike most current clinical laboratory assays of proteins, the approach used does not rely on the specificity of antibodies for capture and detection. Instead, the assay described by Bondar et al. cleaves the protein into small peptides by proteolysis with trypsin. By use of LC-MS/MS, one of the peptide fragments released from zinc-2-glycoprotein is ratioed vs a stable isotope-labeled form of the same peptide. The method serves as an example of an approach to protein quantification that does not require specific antibody reagents and may be broadly applicable.
The instrument used for this analysis, an LC-triple quadrupole mass spectrometer, is an increasingly common tool in the clinical laboratory. Its range of applications now includes routine assays for (a) detecting inborn errors of metabolism, (b) monitoring therapeutic drugs, and (c) quantifying steroid and thyroid hormones. The technology provides high selectivity and high throughput in a clinical laboratory environment. MS/MS usually offers high specificity, even in complex sample matrices, through selection of a specific precursor ion in the first mass analyzer and selection of a specific fragment ion formed during passage of the precursor ion through a collision cell. Experience analyzing therapeutic drugs and hormones in blood shows the ability of the method to measure components down to nanomole per liter concentrations. Considering that each of the 30 most abundant plasma proteins usually has concentrations well over 1 µmol/L (2), many proteins are likely to be sufficiently abundant for analysis by this technique in unfractionated specimens. Low abundance proteins, with concentrations <1 nmol/L, however, are likely to require some form of fractionation or concentration, such as immunocapture of the target protein or peptide (3).
The report by Bondar et al. (1) is but one of numerous reports describing the application of LC-MS/MS to the quantification of proteins for research purposes. Most of these studies have described approaches for relative quantification by use of differential isotopic labeling of paired specimens (4). A number of reports describe the absolute quantification of proteins through use of stable isotope-labeled internal standard peptides (3)(5)(6)(7)(8). Such internal standards can readily be prepared, either by chemical synthesis or by in vitro translation. Most of the applications developed in research laboratories, however, have relied on capillary LC, nanospray interfaces, and ion trap or other types of mass spectrometers not commonly found in clinical laboratories (3)(4)(6)(7)(8). These LC-MS/MS techniques may be more challenging to apply as routine analytical techniques than the LC-MS/MS approaches commonly applied in clinical laboratories. Although the report by Bondar et al. (1) conceptually does not break new ground in its analytical approach, it does illustrate how quantitative analysis of proteins by LC-MS/MS is becoming a practical technique for clinical laboratories to implement, using equipment that currently is applied to other clinical analyses.
There has been some question about how new technologies developed for proteomic analysis will impact the clinical laboratory (2)(9)(10)(11). Profiling of intact peptides or proteins by 2-dimensional electrophoresis or matrix- assisted laser desorption time-of-flight mass spectrometers serves as a discovery tool for top-down proteomics. Chromatographic separations of proteolytic digests of proteins analyzed by ion trap or Fourier-transform ion cyclotron resonance mass spectrometers offer approaches for bottom-up proteomics. These approaches, however, present substantial challenges for routine clinical application and for achieving the usual standards of clinical laboratory practice (12). In contrast, multiple reaction monitoring in LC-MS/MS, using triple-quadrupole mass spectrometers, is a proven clinical laboratory technique for quantitative analysis of molecules in complex matrices such as serum and plasma. Application of this technique to protein analysis shows promise for providing high accuracy and analytical specificity, and should be readily adaptable to simultaneous multiplex analysis of many proteins. The one major limitation of this technique, from the standpoint of proteomic analysis, is that it is a targeted approach, which requires foreknowledge of which proteins to analyze. This characteristic can be a drawback from the standpoint of marker discovery, but not necessarily from the standpoint of a clinical assay, for which analytes preferably are known entities.
It is becoming increasingly evident that new technologies evolving from proteomics research are not simply of academic or research interest. LC-MS/MS employing multiple reaction monitoring is one example of a new technology for protein analysis that is likely to have substantial practical impact in clinical laboratories. One of the most immediate consequences of the application of this technology is the development of new reference methods to standardize protein assays. Two recent examples are restandardization of laboratory measures of hemoglobin A1c (13) and C-peptide (14). This approach is likely to be applied progressively to standardization of a wider range of protein assays, such as for urine albumin using a recently described LC-MS/MS technique (15). Because LC-MS/MS measures the abundance of only one peptide segment of a protein, expression of the abundance of this peptide in units of molarity rather than mass/volume generally is more appropriate, and it may be possible to develop chemically-synthesized peptides that can serve as reference materials for standardization of protein assays. Finally, the ability to measure the concentration of any protein of sufficient abundance, without the need for purified reference preparations or specific antibodies, should accelerate the discovery and application of measurements of additional proteins for diagnostic applications.
References
2-glycoprotein (ZAG) by LC-MS/MS: a potential serum biomarker for prostate cancer. Clin Chem 2007;53:673-678.The following articles in journals at HighWire Press have cited this article:
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  G. L. Hortin, D. Sviridov, and N. L. Anderson High-Abundance Polypeptides of the Human Plasma Proteome Comprising the Top 4 Logs of Polypeptide Abundance Clin. Chem., October 1, 2008; 54(10): 1608 - 1616. [Abstract] [Full Text] [PDF]
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Y. A. Daniel, C. Turner, R. M. Haynes, B. J. Hunt, and R. N. Dalton Quantification of Hemoglobin A2 by Tandem Mass Spectrometry Clin. Chem., August 1, 2007; 53(8): 1448 - 1454. [Abstract] [Full Text] [PDF]
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R. L. Gundry and J. E. Van Eyk Unraveling the Complexity of Circulating Forms of Brain Natriuretic Peptide Clin. Chem., July 1, 2007; 53(7): 1181 - 1182. [Full Text] [PDF]
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