Chad Borges (Presenter)
Arizona State University
Bio: Chad Borges, Ph.D., is an Assistant Professor with joint appointments in the School of Molecular Sciences and The Biodesign Institute at Arizona State University (ASU). His research interests reside in biomedical applications of mass spectrometry with specific projects focusing on glycans as cancer markers, development of forensic-like markers of biospecimen integrity, and characterization of protein posttranslational modifications as indicators of disease and physiological function. Prior to moving to ASU, Dr. Borges helped establish the Olympic-level certified Sports Medicine Research & Testing Laboratory in Salt Lake City, Utah.
Authorship: Chad R. Borges, Joshua W. Jeffs, Shadi Ferdosi
Arizona State University
| Short Abstract Every year, improprieties in the pre-analytical handling & storage of blood plasma/serum (P/S) specimens employed in clinical laboratory research produce false leads--particularly in the world of biomarker discovery. Temporary exposure to the thawed state (> -30°C) is difficult if not impossible to comprehensively track. We have developed a low sample-volume (≤10 µL), dilute-and-shoot, intact protein mass spectrometric assay of albumin proteoforms, “ΔS-Cys-Albumin”, that estimates the equivalent number of room temperatures hours to which stored P/S samples have been exposed over the course of their lifetimes. The assay is based on the known population reference ranges for albumin, cystine, free cysteine and adventitious Cu2+ in P/S used in conjunction with our newly determined multiple-reaction rate law that accurately predicts the ex vivo formation of S-Cys-Albumin over time. |
Long Abstract
Introduction
Every year, improprieties in the pre-analytical handling and storage of blood plasma/serum (P/S) specimens generate false leads in mass spectrometry-based biomedical research that aims to develop clinical applications. Considering the entire life of a typical research specimen, temporary exposure to the thawed state (i.e., any temperature > -30°C) is not uncommon--yet, arguably, no other pre-analytical variable is more difficult to fully control and comprehensively track. Years of experience with the analysis of intact proteins by mass spectrometry has led us to realize the ubiquitous nature of protein oxidation as a mechanism of ex vivo protein instability. As such, we sought to capitalize on this phenomenon as a means of systematically tracking and evaluating P/S specimen integrity.
In short, we have developed a simple, low volume-consumption (≤ 10 µL) assay to estimate the lifetime exposure of P/S samples to the thawed state based on two separate measurements of the relative fractional abundance of S-cysteinylated albumin (S-Cys-Albumin) that 1) is based on the fact that the relative abundance of S-Cys-Albumin will always increase over time (but to a maximum value) when fresh P/S is handled/stored above its melting point of -30°C, 2) is not impacted by in vivo oxidative stress, and 3) is based on the known population reference ranges for albumin, cystine, free cysteine and adventitious Cu2+ in P/S used in conjunction with our newly determined multiple-reaction rate law that accurately predicts the ex vivo formation of S-Cys Albumin over time.
In addition to describing the theoretical underpinnings, development and analytical performance characteristics of this “ΔS-Cys-Albumin” assay, we will present a case study of a “real life” set of samples that were collected by highly respected, NIH-funded investigators and accompanied by a pristine paper trail--but in which ΔS-Cys-Albumin detected and forced exposure of a previously undisclosed sample integrity problem.
Methods
We have published our LC/MS method for the measurement of S-Cys-Albumin and the fact that it increases when P/S are exposed to temperatures > -30°C [1]. Briefly, immediately upon thawing, 0.5 µL of P/S is diluted into 500 µL of 0.1% trifluoroacetic acid (TFA) and 5 µL of diluted sample is injected onto an LC-MS configured with a simple reversed-phase polymeric protein captrap (instead of a column), operating in full scan mode and configured for analysis of intact proteins. To measure ΔS-Cys-Albumin, an additional 9.5 µL of P/S is transferred to a separate 0.5-mL test tube and incubated at 37 °C for 18 hrs. Subsequently, 0.5 µL is withdrawn from the incubated P/S, diluted 1000x in 0.1% TFA and injected onto the LC-MS (in a second run). Using a step gradient, albumin chromatographically elutes prior to the other readily observable protein, apolipoprotein A-I. Peak heights from charge deconvoluted mass spectra of albumin are employed to determine the relative abundance of reduced and oxidized albumin (Albumin-SH and S-Cys-Albumin, respectively). The difference between the final and initial measurements of the fractional relative abundance of S-Cys-Albumin constitutes ΔS-Cys-Albumin.
Results
Theory and Development: The ΔS-Cys-Albumin test is based on the empirically observed fact that the relative abundance of S-cysteinylated (oxidized) albumin (S-Cys-Albumin or Albumin-S-Cys) in P/S will always increase over time (but to a maximum value) when P/S is handled/stored above its melting point of -30°C [1]. This phenomenon is based on two coupled non-enzymatic redox reactions:
1) Albumin-SH + Cystine <--> Albumin-S-Cys + Cys
2) 4Cys + O2 -> 2Cystine + 2H2O [Cu2+ catalyzed]
Where Albumin-SH is the reduced, single-free-thiol-containing albumin proteoform, cystine is the disulfide dimer of the free amino acid cysteine, Albumin-S-Cys is the oxidized proteoform of albumin (S-Cys-Albumin) and Cys is free cysteine. The ΔS-Cys-Albumin assay is also based on the fact that the population reference ranges for the concentrations of albumin, cystine and cysteine in P/S in vivo are 650 ± 60 (SD) µM, 70 ± 10 (SD) µM, and 11 ± 1 (SD) µM, respectively. To enable accurate interpretation of ΔS-Cys-Albumin measurements, we have recently determined the forward and reverse rate constants for reaction 1 at room temperature. The rate law for reaction 2 (with Cu2+ as the catalyst) at room temperature is known. By combining this information into a single unified rate law, we are now able to accurately predict the concentration and relative abundance of S-Cys-Albumin in serum at any point in time given initial (in vivo) starting concentrations of Albumin-SH, S-Cys-Albumin, cystine, cysteine and adventitious Cu2+.
Our model, combined with the known in vivo reference ranges of the biochemical components, along with numerous empirical observations to date have confirmed the inevitable increase in S-Cys-Albumin to a predictable maximum plateau over time. We have noticed, however, that the initial relative abundance of S-Cys-Albumin can be modestly elevated in vivo under conditions of extreme oxidative stress such as acute coronary syndrome. This makes interpretation of a single measurement of S-Cys-Albumin difficult, if not impossible.
To circumvent this problem, we take advantage of the fact that there are always free cystine and free cysteine equivalents in P/S in vivo--which means that upon intentional ex vivo incubation of P/S samples in the thawed state, there will always be an increase in S-Cys-Albumin in fresh, uncompromised samples. The lack of an increase in a P/S sample with an unknown storage history indicates that the sample must have been exposed to thawed conditions (at some point(s) in its lifetime) for a long enough period of time to hit the maximum plateau in S-Cys-Albumin. Thus by measuring S-Cys-Albumin before and after an intentional incubation period that causes S-Cys-Albumin to hit its maximum value, the difference between these values, ΔS-Cys-Albumin, is readily interpreted as inversely proportional to the degree of ex vivo oxidation that occurred prior to the first measurement of S-Cys-Albumin. Thus, for example, a ΔS-Cys-Albumin value of zero would indicate a badly mistreated sample.
Because the rate law at room temperature is now known, we can create individualized reports for unknown samples that state, “Assuming average initial (in vivo) concentrations of albumin, cystine, Cys and adventitious Cu2+, this sample has been exposed to conditions equivalent to 23 °C for x ± y hours.” Moreover, based on the rate law and known precision of the assay [1], it has 90% power (with n = 3) to detect a statistically significant change in plasma S-Cys-Albumin after only 30 minutes of exposure to room temperature.
Case Study: When applied to a nominally pristine set of stage I lung adenocarcinoma cases and age/gender/smoking matched controls that were collected under the same standard operating procedure (SOP) as part of an NCI-sponsored research program, the ΔS-Cys-Albumin assay revealed a major biospecimen integrity discrepancy between the cases and controls (ROC c-statistic for ΔS-Cys-Albumin of 0.96). Though previously undisclosed, further investigation revealed that the -80°C freezers containing most of the control specimens had experienced a 3-4 day loss of power during a natural disaster. Moreover, as expected, the control samples that experienced the temporary increase in temperature had significantly lower ΔS-Cys-Albumin values than the residual control samples that did not experience the power outage.
Conclusions & Discussion
The simple, low volume-consumption (≤ 10 µL), intact protein mass spectrometry-based ΔS-Cys-Albumin assay is founded on well-established biomolecular reference ranges and our recently determined multi-reaction rate law that accurately predicts its ex vivo behavior. For freshly collected plasma, the assay can detect as little as 30 minutes of exposure to room temperature. Moreover, regardless of the exact set of thawed condition(s) to which a P/S sample has been exposed, measurement of ΔS-Cys-Albumin allows an assessment of the equivalent number of room temperature hours to which the sample was exposed over the course of its lifetime. As proof of its “real life” utility, the ΔS-Cys-Albumin assay was used to identify damaged P/S samples within a highly pedigreed set of specimens for which there had originally been no concern about potential integrity issues.
References & Acknowledgements:
Reference Cited:
1. Borges CR, Rehder DS, Jensen S, Schaab MR, Sherma ND, Yassine H, Nikolova B, Breburda C: Elevated Plasma Albumin and Apolipoprotein A-I Oxidation under Suboptimal Specimen Storage Conditions. Molecular & Cellular Proteomics: 2014, 13(7):1890-1899.
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