Elevated Plasma Albumin and Apolipoprotein A-I Oxidation under Suboptimal Specimen Storage Conditions
2014; Elsevier BV; Volume: 13; Issue: 7 Linguagem: Inglês
10.1074/mcp.m114.038455
ISSN1535-9484
AutoresChad R. Borges, Douglas S. Rehder, Sally E. Jensen, Matthew R. Schaab, Nisha D. Sherma, Hussein N. Yassine, Boriana R. Nikolova, Christian S. Breburda,
Tópico(s)Protein Interaction Studies and Fluorescence Analysis
ResumoS-cysteinylated albumin and methionine-oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC–electrospray ionization–MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at −80 °C, −20 °C, and room temperature and exposed to multiple freeze–thaw cycles and other adverse conditions in order to assess the possibility that protein oxidation may occur as a result of poor sample storage or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly; significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at −20 °C. Methionine oxidation of apoA-I took place on a longer time scale, setting in after albumin oxidation reached a plateau. Freeze–thaw cycles had a minimal effect on protein oxidation. In matched collections, protein oxidation in serum was the same as that in plasma. Albumin and apoA-I oxidation were not affected by sample headspace or the degree to which vials were sealed. ApoA-I, however, was unexpectedly found to oxidize faster in samples with lower surface-area-to-volume ratios. An initial survey of samples from patients with inflammatory conditions normally associated with elevated oxidative stress—including acute myocardial infarction and prostate cancer—demonstrated a lack of detectable apoA-I oxidation. Albumin S-cysteinylation in these samples was consistent with known but relatively brief exposures to temperatures above −30 °C (the freezing point of blood plasma). Given their properties and ease of analysis, these oxidized proteoforms, once fully validated, may represent the first markers of blood plasma specimen integrity based on direct measurement of oxidative molecular damage that can occur under suboptimal storage conditions. S-cysteinylated albumin and methionine-oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC–electrospray ionization–MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at −80 °C, −20 °C, and room temperature and exposed to multiple freeze–thaw cycles and other adverse conditions in order to assess the possibility that protein oxidation may occur as a result of poor sample storage or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly; significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at −20 °C. Methionine oxidation of apoA-I took place on a longer time scale, setting in after albumin oxidation reached a plateau. Freeze–thaw cycles had a minimal effect on protein oxidation. In matched collections, protein oxidation in serum was the same as that in plasma. Albumin and apoA-I oxidation were not affected by sample headspace or the degree to which vials were sealed. ApoA-I, however, was unexpectedly found to oxidize faster in samples with lower surface-area-to-volume ratios. An initial survey of samples from patients with inflammatory conditions normally associated with elevated oxidative stress—including acute myocardial infarction and prostate cancer—demonstrated a lack of detectable apoA-I oxidation. Albumin S-cysteinylation in these samples was consistent with known but relatively brief exposures to temperatures above −30 °C (the freezing point of blood plasma). Given their properties and ease of analysis, these oxidized proteoforms, once fully validated, may represent the first markers of blood plasma specimen integrity based on direct measurement of oxidative molecular damage that can occur under suboptimal storage conditions. Human serum albumin contains a single free cysteine residue (Cys34) that is susceptible to oxidation via disulfide-bond formation with free cysteine amino acids, resulting in S-cysteinylated (oxidized) albumin (1.Peters T. All About Albumin: Biochemistry, Genetics, and Medical Applications. Academic Press, San Diego1996Google Scholar). Human apolipoprotein A-I (apoA-I) 1The abbreviations used are: apoA-I, apolipoprotein A-I; P/S, plasma/serum; sa/vol, surface area to volume; TWO, total weighted oxidation. 1The abbreviations used are: apoA-I, apolipoprotein A-I; P/S, plasma/serum; sa/vol, surface area to volume; TWO, total weighted oxidation. contains three methionine residues (Met86, Met112, and Met148) that can be oxidized to sulfoxides (2.Anantharamaiah G.M. Hughes T.A. Iqbal M. Gawish A. Neame P.J. Medley M.F. Segrest J.P. Effect of oxidation on the properties of apolipoproteins A-I and A-II.J. Lipid Res. 1988; 29: 309-318Abstract Full Text PDF PubMed Google Scholar, 3.von Eckardstein A. Walter M. Holz H. Benninghoven A. Assmann G. Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C-II, and C-III.J. Lipid Res. 1991; 32: 1465-1476Abstract Full Text PDF PubMed Google Scholar, 4.Panzenbock U. 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The effect of storage on the accumulation of oxidative biomarkers in donated packed red blood cells.J. Trauma. 2009; 66: 76-81Crossref PubMed Scopus (28) Google Scholar) and apoA-I (6.Pankhurst G. Wang X.L. Wilcken D.E. Baernthaler G. Panzenbock U. Raftery M. Stocker R. Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein.J. Lipid Res. 2003; 44: 349-355Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) are susceptible to artifactual oxidation ex vivo. Notably, the scientific literature in recent years has been relatively quiet with regard to both of these markers. We suspected that spontaneous artifactual oxidation of these proteins ex vivo led to their initial implication as markers of disease, but that the same phenomenon might have confounded efforts to clinically validate them (11.Woodward M. Croft K.D. Mori T.A. Headlam H. Wang X.S. Suarna C. Raftery M.J. MacMahon S.W. Stocker R. Association between both lipid and protein oxidation and the risk of fatal or non-fatal coronary heart disease in a human population.Clin. Sci. 2009; 116: 53-60Crossref PubMed Scopus (31) Google Scholar, 12.Mullan A. Sattar N. More knocks to the oxidation hypothesis for vascular disease?.Clin. Sci. 2009; 116: 41-43Crossref Scopus (4) Google Scholar). Thus we undertook systematic studies of albumin and apoA-I oxidation ex vivo and found evidence indicating that rather than serving as markers of disease, oxidized albumin and apoA-I may serve as markers for improper handling and storage of blood P/S. Improper biospecimen handling and storage can contribute to sample measurements that do not accurately reflect biological reality in vivo (13.Lippi G. Guidi G.C. Mattiuzzi C. Plebani M. Preanalytical variability: the dark side of the moon in laboratory testing.Clin. Chem. Lab. Med. 2006; 44: 358-365Crossref PubMed Scopus (332) Google Scholar, 14.Betsou F. Barnes R. Burke T. Coppola D. 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This may introduce bias in analytical results, limiting the capacity for meaningful comparisons among patient groups (17.Rai A.J. Gelfand C.A. Haywood B.C. Warunek D.J. Yi J.Z. Schuchard M.D. Mehigh R.J. Cockrill S.L. Scott G.B.I. Tammen H. Schulz-Knappe P. Speicher D.W. Vitzthum F. Haab B.B. Siest G. Chan D.W. HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples.Proteomics. 2005; 5: 3262-3277Crossref PubMed Scopus (467) Google Scholar, 18.Lim M.D. Dickherber A. Compton C.C. Before you analyze a human specimen, think quality, variability, and bias.Anal. Chem. 2011; 83: 8-13Crossref PubMed Scopus (45) Google Scholar, 19.Tuck M.K. Chan D.W. Chia D. Godwin A.K. Grizzle W.E. Krueger K.E. Rom W. Sanda M. Sorbara L. Stass S. Wang W. Brenner D.E. 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Nonetheless, identification and standardization of quality control markers that cover this specific scope of application (i.e. proper storage conditions for blood P/S) represent an important goal of biobanking-related research (16.Betsou F. Gunter E. Clements J. DeSouza Y. Goddard K.A. Guadagni F. Yan W. Skubitz A. Somiari S. Yeadon T. Chuaqui R. Identification of evidence-based biospecimen quality-control tools: a report of the International Society for Biological and Environmental Repositories (ISBER) Biospecimen Science Working Group.J. Mol. Diagn. 2013; 15: 3-16Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 20.LaBaer J. Improving international research with clinical specimens: 5 achievable objectives.J. Proteome Res. 2012; 11: 5592-5601Crossref PubMed Scopus (28) Google Scholar). Betsou et al. (16.Betsou F. Gunter E. Clements J. DeSouza Y. Goddard K.A. Guadagni F. Yan W. Skubitz A. Somiari S. Yeadon T. Chuaqui R. Identification of evidence-based biospecimen quality-control tools: a report of the International Society for Biological and Environmental Repositories (ISBER) Biospecimen Science Working Group.J. Mol. Diagn. 2013; 15: 3-16Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) recently outlined and ranked some of the strongest candidates for use as quality control tools in biomarker research. Within the scope of tools for assessing proper handling and storage of P/S samples, nearly all markers are founded on the quantification of a nominal protein via a molecular-recognition-based assay. As a result, the indication of a loss of specimen integrity lies in an apparent loss of the target protein beyond the normal human reference range. Such loss is often ascribed to "degradation" and in many cases likely happens because of residual proteolytic activity that occurs at temperatures above the sample freezing point. In other cases, loss of the protein marker may be due to misfolding caused by repeated freeze–thaw cycles. Though not frequently discussed, protein "degradation" ex vivo may also have roots in oxidative processes that are capable of disrupting protein–antibody interactions that serve as the basis for protein quantification. It is well known that in the absence of special precautions, disulfide bonds will form spontaneously between cysteine thiols. We have previously shown that this requires only the presence of atmospheric oxygen and trace metals and proceeds through a cysteine sulfenic acid intermediate (21.Rehder D.S. Borges C.R. Cysteine sulfenic acid as an intermediate in disulfide bond formation and nonenzymatic protein folding.Biochemistry. 2010; 49: 7748-7755Crossref PubMed Scopus (109) Google Scholar). This mechanism also applies to S-cysteinylation of albumin (22.Turell L. Botti H. Carballal S. Ferrer-Sueta G. Souza J.M. Duran R. Freeman B.A. Radi R. Alvarez B. 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High-performance liquid chromatography of rat and mouse islet polypeptides: potential risk of oxidation of methionine residues during sample preparation.J. Chromatogr. 1990; 530: 29-37Crossref PubMed Scopus (17) Google Scholar, 26.Brot N. Weissbach H. Biochemistry of methionine sulfoxide residues in proteins.BioFactors. 1991; 3: 91-96PubMed Google Scholar); indeed, artifactual sulfoxidation of methionine residues in peptide-based proteomics work is well known. Oxidative modifications such as these have the potential to disrupt antibody interactions with the oxidized protein, resulting in low readings in molecular-recognition-based assays. Thus protein oxidation merits investigation as a protein "degradation" pathway. As pointed out by Betsou et al. (16.Betsou F. Gunter E. Clements J. DeSouza Y. Goddard K.A. Guadagni F. Yan W. Skubitz A. Somiari S. Yeadon T. Chuaqui R. Identification of evidence-based biospecimen quality-control tools: a report of the International Society for Biological and Environmental Repositories (ISBER) Biospecimen Science Working Group.J. Mol. Diagn. 2013; 15: 3-16Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), markers that are highly sensitive to variations in specimen storage and handling conditions are likely to be the most useful. A considerable degree of change that occurs rapidly under undesirable conditions to which samples may be exposed, such as the state of being incompletely frozen (which for blood plasma occurs at temperatures above −30 °C (27.Farrugia A. Hill R. Douglas S. Karabagias K. Kleinig A. Factor VIII/von Willebrand factor levels in plasma frozen to -30 degrees C in air or halogenated hydrocarbons.Thromb. Res. 1992; 68: 97-102Abstract Full Text PDF PubMed Scopus (8) Google Scholar, 28.MacKenzie, A. 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EDTA blood plasma specimens for time-course studies were collected via forearm venipuncture under institutional review board approval at the University of Southern California, processed within 30 min at room temperature, and immediately frozen at −80 °C. Samples were shipped on dry ice to Arizona State University, where they were received frozen and subsequently aliquoted for their respective time courses at various temperatures. Matched EDTA plasma and serum samples from healthy volunteers were collected at Arizona State University under institutional review board approval. These samples were collected via forearm venipuncture according to NIH's Early Detection Research Network blood collection standard operating procedures (31.The Early Detection Research Network (EDRN) Standard Operating Procedure (SOP) for Collection of EDTA Plasma.Google Scholar, 32.The Early Detection Research Network (EDRN) Standard Operating Procedure (SOP) for Collection of Serum.Google Scholar). Plasma samples were processed at room temperature, aliquoted, and placed in a −80 °C freezer within 35 min of collection; serum samples were placed at −80 °C within 95 min of collection. Blood from patients experiencing acute coronary syndrome symptoms (later confirmed as myocardial infarction) was collected upon patient arrival at the Maricopa Integrated Health Systems emergency room or cardiology clinic. Patients were consented and blood was collected via forearm venipuncture under institutional review board approval. Blood serum was processed at room temperature within 3 h of collection, and samples were stored for 0 to 6 weeks at −70 °C prior to transfer on dry ice to Arizona State University for analysis. Basic patient clinical information and laboratory data are provided in supplemental Tables S6 and S7. Blood serum from prostate cancer patients was purchased from the Cooperative Human Tissue Network (an NIH-supported blood- and tissue-collection bank). Specimens were processed according to their standard protocols. The Cooperative Human Tissue Network stored the samples at −80 °C prior to distribution, when they were shipped on dry ice. Samples were subjected to two freeze–thaw and aliquoting cycles for other purposes prior to analysis for this study. Patient demographics and laboratory data are provided in supplemental Table S8. Plasma and serum samples were thawed at room temperature, mixed by vortexing, and then centrifuged at 13,000g for 1.5 min to sediment any particulates. 0.5 μl was removed and diluted into 500 μl of 0.1% (v/v) trifluoroacetic acid. 5 μl of this solution were injected without delay by a Spark Holland Endurance autosampler in microliter pick-up mode and loaded by an Eksigent nanoLC*1D at 10 μl/min using 80% water/20% acetonitrile with 0.1% formic acid onto a protein captrap (Michrom, city/country no longer commercially available) configured for unidirectional flow on a six-port diverter valve. The captrap was then washed for 3 min with this loading solvent. The flow rate over the protein captrap cartridge was then changed to 1 μl/min, and a linear gradient of increasing acetonitrile from 20% to 90% was employed to elute the proteins into the mass spectrometer. The captrap eluent was directed to a Bruker MicrOTOF-Q (Q-TOF) mass spectrometer operating in positive ion, TOF-only mode, acquiring spectra in the m/z range of 300 to 3000 with a resolving power of ∼20,000 m/Δm full width at half-maximum. Electrospray ionization settings for the Agilent G1385A capillary microflow nebulizer ion source were as follows: end plate offset -500 V, capillary -4500 V, nebulizer nitrogen 2 bar, and dry gas nitrogen 3.0 l/min at 225 °C. Notably, this electrospray ionization mass spectrometer has a source design in which the spray needle is kept at ground and the inlet of the instrument is brought to a high negative voltage (in positive ion mode). This design is important because it avoids the possibility of corona discharge and subsequent artifactual protein oxidation (33.Boys B.L. Kuprowski M.C. Noel J.J. Konermann L. Protein oxidative modifications during electrospray ionization: solution phase electrochemistry or corona discharge-induced radical attack?.Anal. Chem. 2009; 81: 4027-4034Crossref PubMed Scopus (73) Google Scholar). Data were acquired in profile mode at a digitizer sampling rate of 2 GHz. Spectra rate control was by summation at 1 Hz. Approximately 1 min of recorded spectra were averaged across the chromatographic peak apex of each protein of interest. The electrospray ionization charge-state envelope was deconvoluted with Bruker DataAnalysis v3.4 software to a mass range of 1000 Da on either side of any identified peak. Deconvoluted spectra were baseline subtracted, and all peak heights were calculated. Peak heights were employed for quantification instead of area because of the incomplete resolution of some of the peaks involved. Tabulated mass spectral peak heights were exported to a spreadsheet for further calculation. The fractional abundance of S-cysteinylated (oxidized) albumin was determined by dividing the height of the mass spectral peak representing S-cysteinylated albumin by the sum of the peak heights for native and S-cysteinylated albumin. ApoA1 contains three Met residues; thus each molecule of apoA-I may contain no, one, two, or three oxidized Met residues. The relative distribution of apoA-I in each of these four oxidation states can readily be determined via electrospray ionization MS. This degree of analytical clarity affords the expression of a unique measurement of apoA-I oxidation that we refer to as total weighted oxidation (TWO). This term refers to the weighted fractional abundance of maximum Met oxidation (i.e. the case in which all three Met residues of apoA-I are oxidized to sulfoxides). To calculate the TWO or percent total oxidation capacity (34.Yassine H. Borges C.R. Schaab M.R. Billheimer D. Stump C. Reaven P. Lau S.S. Nelson R. Mass spectrometric immunoassay and MRM as targeted MS-based quantitative approaches in biomarker development: potential applications to cardiovascular disease and diabetes.Proteomics Clin. Applicat. 2013; 7: 528-540Crossref PubMed Scopus (43) Google Scholar), the mass spectral peak heights of native (unoxidized) apoA1 and of singly, doubly, and triply oxidized apoA1 are multiplied by 0, 0.33, 0.66, and 1, respectively, before normalizing for total apoA1 peak height. Although the analytical methods employed in this study reproducibly measure the relative abundance of oxidized albumin and apoA-I proteoforms, they are not validated for absolute protein quantification. As a result, no conclusions can be drawn about protein degradation over time based on raw or charge deconvoluted signal intensity. Dilution of blood P/S followed by direct analysis via mass spectrometry allows for the detection of both albumin and apoA-I. Bar-Or and colleagues have described dilute-and-shoot LC-MS methods for the analysis of intact albumin and its S-cysteinylated form (10.Rael L.T. Bar-Or R. Ambruso D.R. Mains C.W. Slone D.S. Craun M.L. Bar-Or D. The effect of storage on the accumulation of oxidative biomarkers in donated packed red blood cells.J. Trauma. 2009; 66: 76-81Crossref PubMed Scopus (28) Google Scholar, 35.Bar-Or D. Bar-Or R. Rael L.T. Gardner D.K. Slone D.S. Craun M.L. Heterogeneity and oxidation status of commercial human albumin preparations in clinical use.Crit. Care Med. 2005; 33: 1638-1641Crossref PubMed Scopus (131) Google Scholar). We found that a similar approach could be used to analyze apoA-I simultaneously. This simple dilute-and-shoot method provided clean, easily interpreted mass spectra for the relative quantification of both albumin and apoA-I oxidation (Fig. 1). Raw spectra and peak tables corresponding to the charge deconvoluted spectra shown in Fig. 1 are provided in supplemental Figs. S1–S5 and supplemental Tables S1–S5. These supplemental figures and tables provide details on raw m/z values, charge states, MH+ protein masses from charge deconvoluted spectra, and typically observed peak heights. They represent data from across the entire qualitative and quantitative ranges of albumin and apoA-I proteoforms observed in these studies (i.e. no additional unique proteoforms of albumin or apoA-I were observed). Peak assignments in charge deconvoluted spectra were made on the basis of absolute mass. The calculated MH+ average mass of unmodified human albumin is 66,438.9 Da, and that of apoA-I is 28,079.6 Da. Based on analytical reproducibility data (described below), the unmodified form of albumin in charge deconvoluted spectra was measured at 66,439.45 ± 1.2 Da (S.E.). Unmodified apoA-I was measured at 28,079.6 ± 0.57 Da (S.E.). All modified forms of albumin (5.Bar-Or D. Heyborne K.D. Bar-Or R. Rael L.T. Winkler J.V. Navot D. Cysteinylation of maternal plasma albumin and its association with intrauterine growth restriction.Prenat. Diagn. 2005; 25: 245-249Crossref PubMed Scopus (47) Google Scholar, 10.Rael L.T. Bar-Or R. Ambruso D.R. Mains C.W. Slone D.S. Craun M.L. Bar-Or D. The effect of storage on the accumulation of oxidative biomarkers in donated packed red blood cells.J. Trauma. 2009; 66: 76-81Crossref PubMed Scopus (28) Google Scholar, 35.Bar-Or D. Bar-Or R. Rael L.T. Gardner D.K. Slone D.S. Craun M.L. Heterogeneity and oxidation status of commercial human albumin preparations in clinical use.Crit. Care Med. 2005; 33: 1638-1641Crossref PubMed Scopus (131) Google Scholar) and apoA-I (6.Pankhurst G. Wang X.L. Wilcken D.E. Baernthaler G. Panzenbock U. Raftery M. Stocker R. Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein.J. Lipid Res. 2003; 44: 349-355Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) have been described and published elsewhere and are in agreement with the modifications due to mild oxidation reported in these studies. Reproducibility and autosampler stability were validated to ensure the integrity of stability study results. Intra- and interday reproducibility of the analytical method were evaluated. For albumin, samples from three males and three females were analyzed in quadruplicate on three separate days. The average fractional abundance of S-cysteinylated albumin in these samples was 0.25. The average within-day precision (expressed as %cv) was 5.2%, and the total interday precision was 6.2%. ApoA-I oxidation was not detected in any of these samples. Data on the reproducibility of the apoA-I oxidation assay were thus collected from a separ
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