...LC-MS/MS-based clinical protein analysis has predominantly focused on improved analytical measurement for well-established biomarkers (5). Postextraction stability should be determined for both ...pools after storage in the autosampler (^24 h, reinjecting aliquots if feasible), freezing (^72 h, if rou- tine), and extract freeze-thaw for 1 and 2 cycles. Because many preclinical studies rely on biobanked materials, it should be noted that at least 3 freshly acquired samples should be evaluated for stability of 1 freeze-thaw cycle (assay fresh, freeze for ^12 h, thaw for ^2 h, reassay, and compare).
The utilization of vaccines to fight the spread of SARS-CoV-2 has led to a growing need for expansive serological testing. To address this, an EUA approved immunoassay for detection of antibodies to ...SARS-CoV-2 in venous serum samples was investigated for use with dried blood spot (DBS) samples. Results from self-collected DBS samples demonstrated a 98.1% categorical agreement to venous serum with a correlation (R) of 0.9600 while professionally collected DBS samples demonstrated a categorical agreement of 100.0% with a correlation of 0.9888 to venous serum. Additional studies were performed to stress different aspects of at-home DBS collection, including shipping stability, effects of interferences, and other sample-specific robustness studies. These studies demonstrated a categorical agreement of at least 95.0% and a mean bias less than ± 20.0%. Furthermore, the ability to track antibody levels following vaccination with the BioNTech/Pfizer vaccine was demonstrated with serial self-collected DBS samples from pre-dose (Day 0) out to 19 weeks.
In the phase III study COU-AA-301, abiraterone acetate (AA) plus prednisone (P) prolonged overall survival (OS) in patients with metastatic castration-resistant prostate cancer (mCRPC) after ...docetaxel administration. In this article, we investigate the relationship between baseline serum androgen (SA) levels and OS.
COU-AA-301 is a randomized, double-blind study of AA (1,000 mg every day) plus P (5 mg by mouth twice daily; n = 797) versus P alone (n = 398). Randomization was stratified by Eastern Cooperative Oncology Group performance status (0 to 1 v 2), pain (Brief Pain Inventory-Short Form over past 24 hours: 4 to 10, present; v 0 to 3, absent), prior chemotherapy (1 v 2), and progression (prostate-specific antigen v radiographic). Association of baseline SA (testosterone, androstenedione, dehydroepiandrosterone sulfate), was measured by ultrasensitive liquid-liquid extraction or protein precipitation and two-dimensional liquid chromatography coupled to mass spectrometry, with OS determined by bivariate and multivariable Cox models. OS was examined with SA as greater than median and less than or equal to the median.
Median survival increased with each quartile increase in testosterone level regardless of treatment arm. SA levels at baseline strongly associated with survival (P < .0001) in bivariate and multivariable analyses. Longer survival was observed for patients with SA above median compared with below median in both the AA and P arms (eg, testosterone, AA; hazard ratio, 0.64; 95% CI, 0.53 to 0.77; P < .0001). Treatment with AA led to longer survival versus P alone in the above- or below-median group for all androgens.
SA, measured with a novel ultrasensitive assay in COU-AA-301, is prognostic for OS and may be useful for risk stratification in mCRPC clinical trials.
Chronic obstructive pulmonary disease (COPD) occurs in a minority of smokers and is characterized by intermittent exacerbations and clinical subphenotypes such as emphysema and chronic bronchitis. ...Although sphingolipids as a class are implicated in the pathogenesis of COPD, the particular sphingolipid species associated with COPD subphenotypes remain unknown.
To use mass spectrometry to determine which plasma sphingolipids are associated with subphenotypes of COPD.
One hundred twenty-nine current and former smokers from the COPDGene cohort had 69 distinct sphingolipid species detected in plasma by targeted mass spectrometry. Of these, 23 were also measured in 131 plasma samples (117 independent subjects) using an untargeted platform in an independent laboratory. Regression analysis with adjustment for clinical covariates, correction for false discovery rate, and metaanalysis were used to test associations between COPD subphenotypes and sphingolipids. Peripheral blood mononuclear cells were used to test associations between sphingolipid gene expression and plasma sphingolipids.
Of the measured plasma sphingolipids, five sphingomyelins were associated with emphysema; four trihexosylceramides and three dihexosylceramides were associated with COPD exacerbations. Three sphingolipids were strongly associated with sphingolipid gene expression, and 15 sphingolipid gene/metabolite pairs were differentially regulated between COPD cases and control subjects.
There is evidence of systemic dysregulation of sphingolipid metabolism in patients with COPD. Subphenotyping suggests that sphingomyelins are strongly associated with emphysema and glycosphingolipids are associated with COPD exacerbations.
Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful ...application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this “fit-for-purpose” approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.
Stable isotopically labeled (SIL) tryptic peptides, cleavable SIL peptides, and a full-length SIL protein were compared for internal calibration (i.e., as internal calibrators) and external ...calibration (i.e., as internal standards) when quantifying three forms of unlabeled, human thyroglobulin (Tg) by bottom-up protein analysis. All SIL materials and human proteins were standardized by amino acid analysis to ensure traceability of measurements and allow confident assignment of accuracy. The three forms of human Tg quantified were (1) the primary reference material BCR457a native protein purified from human thyroids, (2) a commercially available form also purified from human thyroids, and (3) a full-length recombinant form expressed and purified from a human embryonic kidney 293 cell-line. Collectively, the results unequivocally demonstrate the lack of commutability of tryptic and cleavable SIL peptides as internal calibrators across various bottom-up assays (i.e., denaturing/digestion conditions). Further, the results demonstrate the potential during external calibration for surrogate protein calibrators (i.e., recombinant proteins) to produce inaccurate concentration assignments of native protein analytes by bottom-up analysis due to variance in digestion efficiency, which is not alleviated by altering denaturation/digestion stringency and indicates why protein calibrators may not be commutable in bottom-up protein assays. These results have implications regarding the veracity of “absolute” protein concentration assignments by bottom-up assays using peptide calibrators, as well as protein calibrators, given that absolute accuracy was not universally observed. Nevertheless, these results support the use of recombinant SIL proteins as internal standards over SIL peptides due to their ability to better mimic the digestion of human-derived proteins and mitigate bias due to digestion-based matrix effects that were observed during external calibration.
LC-MS-based methods for protein quantification have a stigma of being relatively expensive and low-throughput. This is partly due to the cost and speed of trypsin digestion, which has primarily ...focused on advancements in research-based biomarker discovery applications that rely on protein/peptide identifications rather than clinical biomarker quantification. However, there is a need for simple, fast, and reproducibly efficient surrogate peptide recovery in clinical biomarker quantification.
Multiple methodologies were evaluated to enhance tryptic digestion for the analysis of thyroglobulin, a prototypical serum protein biomarker. The main criteria for assessment were the yield and speed of formation of surrogate peptides. Various factors such as different additives, types of trypsin, microwave- and pressure-assisted systems, and enzyme concentration were considered as key variables, in addition to digestion time.
It was observed that digestion additives/denaturants had a significant impact on the speed and yield of digestion for each surrogate peptide. Increasing the concentration of trypsin alone was found to accelerate digestions appreciably for most surrogate peptides, without affecting the yield. However, the use of sequencing-grade trypsins and microwave/pressure-assisted systems did not offer significant advantages over the use of 'standard-grade' TPCK-treated trypsin in combination with a conventional incubator, once digestion time and additive had been optimized.
We have dispelled the notion that trypsin digestion is inherently slow and expensive for targeted quantification of serum proteins. Additionally, we have established a groundwork for experimentation that can pave the way for the creation of efficient trypsin digestion protocols, aiming to optimize yield, speed, and cost. It is our hope that these advancements will promote the wider incorporation of such assays in clinical laboratories.
Plasma branched-chain amino acid (BCAA) levels, measured on nuclear magnetic resonance (NMR) metabolomics research platforms or by mass spectrometry, have been shown to be associated with type 2 ...diabetes mellitus (T2DM) and cardiovascular disease (CVD). We developed a new test for quantification of BCAA on a clinical NMR analyzer and used this test to determine the clinical correlates of BCAA in 2 independent cohorts.
The performance of the NMR-based BCAA assay was evaluated. A method comparison study was performed with mass spectrometry (LC-MS/MS). Plasma BCAA were measured in the Insulin Resistance Atherosclerosis Study (IRAS, n = 1209; 376 T2DM subjects) and in a Groningen cohort (n = 123; 67 T2DM subjects). In addition, carotid intima media thickness (cIMT) was measured successfully in 119 subjects from the Groningen cohort.
NMR-based BCAA assay results were linear over a range of concentrations. Coefficients of variation for inter- and intra-assay precision ranged from 1.8–6.0, 1.7–5.4, 4.4–9.1, and 8.8–21.3%, for total BCAA, valine, leucine, and isoleucine, respectively. BCAA quantified from the same samples using NMR and LC-MS/MS were highly correlated (R2 = 0.97, 0.95 and 0.90 for valine, leucine and isoleucine). In both cohorts total and individual BCAA were elevated in T2DM (P = 0.01 to ≤0.001). Moreover, cIMT was associated with BCAA independent of age, sex, T2DM and metabolic syndrome (MetS) categorization or alternatively of individual MetS components.
BCAA levels, measured by NMR in the clinical laboratory, are elevated in T2DM and may be associated with cIMT, a proxy of subclinical atherosclerosis.
•A novel NMR-based BCAA assay was developed for use in the clinical laboratory.•The clinical NMR BCAA assay exhibits good performance characteristics.•BCAA quantified using both NMR and LC-MS/MS were highly correlated.•High NMR-measured BCAA levels relate to T2DM and MetS in 2 independent cohorts.•cIMT was associated with BCAA independent of age, sex, T2DM and MetS categorization.
The march of the masses Grant, Russell P
Clinical chemistry (Baltimore, Md.)
59, Številka:
6
Journal Article
Recenzirano
Odprti dostop
...the tools and technologies being de- ployed will require further refinement for mass spec- trometers to become established automated clinical analyzers. ...mul- ticoncentration calibrators ...defining the analytical range, often bracketing QCs and samples, control for instrument response drift and deviations from detec- tion linearity over the course of analysis.
Objectives
To review urinary protein biomarkers as potential non‐invasive, easily obtainable, early diagnostic tools in renal cell carcinoma (RCC).
Methods
A PubMed database search was performed up ...to the year 2020 to identify primary studies reporting potential urinary protein biomarkers for RCC. Separate searches were conducted to identify studies describing appropriate methods of developing cancer screening programmes and detection of cancer biomarkers.
Results
Several urinary protein biomarkers are under validation for RCC diagnostics, e.g. aquaporin‐1, perilipin‐2, carbonic anhydrase‐9, Raf‐kinase inhibitory protein, nuclear matrix protein‐22, 14‐3‐3 Protein β/α and neutrophil gelatinase‐associated lipocalin. However, none has yet been validated or approved for clinical use due to low sensitivity or specificity, inconsistencies in appropriate study design, or lack of external validation.
Conclusions
Evaluation of biomarkers’ feasibility, sample preparation and storage, biomarker validation, and the application of novel technologies may provide a solution that maximises the potential for a truly non‐invasive biomarker in early RCC diagnostics.
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