Peptide lost and found: internal standards and the mass spectrometric quantification of peptides.
Clinical chemists are careful to evaluate these and other preanalytical variables that may affect assay results for their patients. Unfortunately, there is often no way to tell if something has gone unexpectedly wrong with sample collection, storage, or preparation until it is too late and a result is already in a medical record being interpreted by a care provider. It would be highly desirable to have an easy way to tell if a sample has been compromised in a way that would effectively eliminate the possibility of accurate protein quantification, but this option will never be possible for the traditional automated immunochemical platforms that serve as the mainstay in clinical laboratories.
In addition to the preanalytical issues with clinical assays, a variety of analytical problems can also complicate the analysis of a biomarker in human samples. For example, lipemia and hyperproteinemia can interfere with the indirect potentiometric measurement of sodium (3). In addition, interconversion of folate metabolites from lysed erythrocytes occurs during the deconjugation reaction before certain chromatographic assays (4).
In this issue of Clinical Chemistry, Bystrom et al. (5) describe taking advantage of the multichannel nature of the tandem mass spectrometer and and including an internal standard to monitor for a surprising analyte-degradation phenomenon that takes place during analysis. As a part of the evaluation of hypertension, plasma renin activity has historically been measured by incubating serum at 37[degrees]C and measuring the conversion of endogenous angiotensinogen to angiotensin I over time. The authors report a novel liquid chromatography-tandem mass spectrometric assay to replace the RIAs already available. In the process, they provide further evidence of an essential mechanism of variation in proteomic assays of human plasma (6).
During the clinical validation of their assay, the authors noticed many samples in which the amount of angiotensin I measured with the current RIA after an 18-h incubation was significantly lower than that measured with their mass spectrometric assay after a 3-h incubation. In addition, samples that had been incubated for 18 h rather than 3 h had significantly lower concentrations of angiotensin I as measured by mass spectrometry. These findings seemed to indicate degradation of the analyte during incubation.
Liquid chromatography-tandem mass spectrometry is an extremely specific methodology. With the selection of the optimal mobile and stationary phases, the retention time, the m/z of the intact molecular ion, and the m z values of fragment ions formed from collision-induced dissociation are characteristic for the molecule of interest. Solutes that coelute from the chromatographic column can suppress the ionization of the analyte (7). To help compensate for any ion suppression that may be present, one can use stable isotope-labeled analogs as internal standards, because they are subjected to the same amount of ion suppression but have different masses that the mass spectrometer can easily distinguish.
In their new plasma renin activity assay, Bystrom et al. (5) actually included 2 internal standard peptides, each with a different mass. One was added at the end of the angiotensin I-generation step to help control for ion suppression. Another was added to the sample before angiotensin I generation to monitor for degradation of the analyte as it was being formed. With this approach, it became apparent that all samples had a slow intrinsic rate of degradation of the internal standard peptide over the 3-h reaction time; however, approximately 2% of the samples had a much more rapid rate of degradation, which led to a greatly reduced final concentration of angiotensin I at the end of the reaction.
In the assay, plasma renin activity was measured by comparing the analyte response ofthe sample to the analyte responses of a calibration curve. The response was defined as the peak area of the endogenous angiotensin I peptide divided by the peak area of the internal standard peptide that was added at the end ofthe incubation step. Of note is that adding the peptide at the beginning of the incubation also allows one to control for both peptide degradation and ion suppression, but this approach might be complicated by variation in rates of angiotensin I accumulation and degradation from sample to sample. Inclusion of a second internal standard peptide of a different mass to monitor for analyte degradation is a clever approach to provide the sample-to-sample QC that is possible only with mass spectrometric methods (and not with immunoassays). Indeed, as evidenced by this careful study, it appears that approximately 2% of all samples previously analyzed by RIA had reported plasma renin activities that were falsely low, especially after an 18-h incubation step.
The intrinsic degradation of peptides is a problem that could plague the field of proteomics and the quantification of proteins by mass spectrometry (6). In the basic-research arena, particularly when immunoassays for the analytes of interest are not available, mass spectrometry can be used in an unbiased fashion to identify and quantify as many proteins in the sample as possible, or in a targeted fashion to quantify only the proteins of interest. Whereas the plasma renin activity assay exploits the endogenous proteolytic activity of renin, unbiased and targeted proteomics assays use exogenous proteinases to degrade proteins into peptides, which then act as surrogates for the intact proteins. Quantitative targeted proteomics assays use stable isotope-labeled internal standard peptides that are spiked into the sample after proteolysis to normalize for ion suppression. The method has been demonstrated to work well for certain proteins in clinical samples, with good correlations to accepted clinical assays (8-10); however, if specific peptides are degraded after they are generated--and it is very likely that some are--spiking an internal standard after proteolysis may not be enough.
Nonspecific degradation of the peptides of interest during the proteolytic digestion of plasma is not the only analytical factor affecting the mass spectrometric quantification of proteins in clinical samples. Matters are further complicated by the variation in the proteolytic digestion of plasma itself from sample to sample and from laboratory to laboratory (11). It is possible that the inclusion of a cleavable stable isotope-labeled internal standard peptide, stable isotope-labeled internal standard polypeptide (12), or stable isotope-labeled protein analog (13) can help control for both proteolytic variability and peptide degradation, but these approaches have not yet been extensively studied in human plasma.
As the authors have pointed out, the proteolytic degradation of the angiotensin I peptide could be caused by an in vivo phenomenon or a process instigated ex vivo. Similar to the use of internal standards to monitor for the analytical variables that complicate mass spectrometric assays, the inclusion of internal standards in blood draw tubes to monitor for certain preanalytical as well as analytical factors that could threaten accuracy is theoretically possible. Unfortunately, the approach is prohibitively expensive at this time.
The goal of many investigators is to use mass spectrometry to precisely quantify proteins in human serum and plasma. The investigation by Bystrom et al. (5) has demonstrated an important potential source of variation that is not detectable unless an internal standard is included at the outset of digestion. Of course, most types of analytical issues are completely masked in immunoassays (14), a fact that makes mass spectrometers exciting new platforms for the measurement of proteins in human plasma.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: A.N. Hoofnagle, Thermo Fisher Scientific.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: A.N. Hoofnagle, Waters and Bruker Daltonics.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
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Andrew N. Hoofnagle *
Department of Laboratory Medicine, University of Washington, Seattle, WA.
* Address correspondence to the author at: Department of Laboratory Medicine, University of Washington, Seattle, WA 98195-7110. Fax 206-598-6189; e-mail firstname.lastname@example.org.
Received July 19, 2010; accepted July 27, 2010.
Previously published online at DOI: 10.1373/clinchem.2010.152181
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|Author:||Hoofnagle, Andrew N.|
|Date:||Oct 1, 2010|
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