BACKGROUND: Quantification of serum tumor markers plays an important role in determining whether patients treated for cancer require further therapy. Whereas large-scale proteomic efforts aim to identify novel tumor markers to facilitate early detection, optimization of methods for quantifying known tumor markers offers another approach to improving management of malignancies. For example, immunoassays used in clinical practice to measure established tumor markers suffer from potential interference from endogenous immunoglobulins and imperfect concordance across platforms-problems that also plague many other immunoassays. To address these important limitations, this study used peptide immunoaffinity enrichment in concert with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify thyroglobulin, a well-characterized tumor marker.

Background Mass spectrometric assays have the potential to replace protein immunoassays in basic science, clinical research, and clinical care. Previous studies have demonstrated the utility of assays using multiple-reaction monitoring mass spectrometry (MRM-MS) for the quantification of proteins in biological samples and many examples of the accuracy of these approaches to quantify spiked analytes have been reported. However, a direct comparison of multiplexed assays using liquid chromatography-tandem mass spectrometry with established immunoassays to measure endogenous proteins has not been reported. Methods We purified the HDL from the plasma of 30 human subjects enrolled in a clinical nutrition research study and used label-free shotgun proteomics approaches to analyze each sample. We then developed two different 6-plex assays that used isotope dilution MRM-MS: one assay used stable isotope labeled peptides and the other used stable isotope labeled apolipoprotein A-I (apoA-I), the most abundant protein in HDL, as internal standards to control for matrix effects and mass spectrometer performance. The shotgun and MRM-MS assays were then compared with commercially available immunoassays for each of the six analytes. Results Quantification by shotgun proteomics approaches correlated poorly with the six protein immunoassays. However, the MRM-MS approaches that used internal standard peptide or a single internal standard protein correlated well. In addition, MRM-MS approaches had good repeatability (<10% CV) and linearity. Conclusions Multiplexed MRM-MS assays correlate well with immunochemical measurements and have acceptable operating characteristics in complex samples. Our results support the proposal that MRM-MS could be used to replace immunoassays in a variety of settings.

BACKGROUND:Insulin-like growth factor 1 (IGF-1) 7 is a key mediator of growth hormone (GH) action and a well-characterized biomarker of GH abuse. Current immunoassays for IGF-1 suffer from poor concordance between platforms, which makes comparison of results between laboratories difficult. Although previous work has demonstrated good interlaboratory imprecision of LC-MS/MS methods when plasma is supplemented with purified proteins, the interlaboratory imprecision of an endogenous protein in the nanogram-per-milliliter concentration range has not been reported.

Objective-To determine whether obesity and insulin resistance associate with changes in the protein content of high-density lipoprotein (HDL) in 2 different groups of men by using targeted proteomics. Methods and Results-Insulin resistance and obesity are hallmarks of type 2 diabetes mellitus and the metabolic syndrome, which confer an increased risk of cardiovascular disease. Recent studies suggest that the protein cargo of HDL makes important contributions to the lipoprotein's cardioprotective effects. In a discovery study, we used isotope dilution mass spectrometry to quantify the relative concentrations of 5 proteins previously implicated in HDL's cardioprotective effects in 3 groups of healthy subjects: lean insulin-sensitive, lean insulin-resistant, and obese insulin-resistant individuals. We validated our findings in a different group of subjects. The clusterin concentration in HDL strongly and negatively associated with insulin resistance and body mass index in both populations. HDL clusterin levels were lower in subjects with low HDL and high triglycerides, key components of the metabolic syndrome. There was an inverse correlation between clusterin levels in HDL and very-low-density lipoprotein/ low-density lipoprotein. Key Words: high-density lipoprotein Ⅲ atherosclerosis Ⅲ apolipoprotein J Ⅲ clusterin Ⅲ intra-abdominal fat Ⅲ insulin resistance Ⅲ obesity M any environmental and genetic factors contribute to the metabolic syndrome, whose hallmark is reduced insulin sensitivity accompanied by hypertension, hypertriglyceridemia, and obesity. 1,2 Obesity and insulin resistance are also components of type 2 diabetes mellitus. 3,4 These factors, in turn, contribute to the initiation and progression of atherosclerotic cardiovascular disease (CVD). 3,4 Another cardinal feature of metabolic syndrome is a low level of high-density lipoprotein (HDL), which strongly associates with an increased risk of atherosclerotic vascular disease. Conclusion-ClusterinOne important cardioprotective function of HDL is to remove cholesterol from cholesteryl ester-laden macrophages in the artery wall. [5][6][7] The anti-inflammatory properties of HDL may also contribute to its antiatherogenic effects. 8,9 HDL's cardioprotective ability may depend on the types of particles generated metabolically and on the fact that HDL in humans with established CVD is dysfunctional. 8 -12 Indeed, animal studies 13-15 convincingly demonstrate that changes in HDL's protein composition can promote atherosclerosis, even when plasma levels of HDL cholesterol (HDL-C) are normal or elevated.To explore the potential clinical relevance of protein composition in HDL's cardioprotective effects, we used targeted proteomics to determine the relationship between the relative concentration of 5 different proteins in HDL and 2 key features of metabolic syndrome: obesity and insulin resistance. We quantified proteins that have previously been implicated in HDL's cardioprotective effects: apolipoprotein (apo) A-I, 6,7 clusterin, 5,16 -20 apoE, 21 apoM, 22,23 ...

BackgroundThe secretion of organic solutes by the proximal tubules is an essential intrinsic kidney function. However, the clinical significance of the kidney’s clearance of tubular secretory solutes is uncertain.MethodsIn this prospective cohort study, we evaluated 3416 participants with CKD from the Chronic Renal Insufficiency Cohort (CRIC) study. We measured plasma and 24-hour urine concentrations of endogenous candidate secretory solutes at baseline, using targeted liquid chromatography–tandem mass spectrometry. The study defined CKD progression by a ≥50% decline in the eGFR, initiation of maintenance dialysis, or kidney transplantation. We used Cox proportional hazards regression to test associations of secretory-solute clearances with CKD progression and mortality, adjusting for eGFR, albuminuria, and other confounding characteristics.ResultsParticipants in this ancillary study had a mean age of 58 years and 41% were black; the median eGFR was 43 ml/min per 1.73 m2. After adjustment, lower kidney clearances of six solutes—kynurenic acid, pyridoxic acid, indoxyl sulfate, xanthosine, isovalerylglycine, and cinnamoylglycine—were associated with significantly greater risks of CKD progression, with clearance of kynurenic acid, a highly protein-bound solute, having the strongest association. Lower clearances of isovalerylglycine, tiglylglycine, hippurate, and trimethyluric acid were significantly associated with all-cause mortality after adjustment.ConclusionsWe found lower kidney clearances of endogenous secretory solutes to be associated with CKD progression and all-cause mortality, independent of eGFR and albuminuria. This suggests that tubular clearance of secretory solutes provides additional information about kidney health beyond measurements of glomerular function alone.

For decades, immunoassays have provided the framework for protein biomarker studies in clinical medicine and in therapeutic monitoring for drug development. At the same time, investigators have uncovered many issues that make immunoassays unreliable in many human serum and plasma samples. LC-MS/MS after tryptic digestion of proteins is potentially an attractive solution, but the sensitivity of the method is not sufficient to measure many important low-abundance proteins directly. The use of antipeptide antibodies to immunoenrich peptides of interest can improve the sensitivity of the approach, greatly simplify the matrix enabling shortened chromatographic runs, and facilitate the multiplexed quantification of analytes, which could reduce the costs of quantitative protein measurements in complex specimens. We provide an overview of the method and the steps needed to develop an assay. In addition, we review the efforts to make this method generally more applicable.

Patients with autoimmune diseases have a significantly increased risk of developing cardiovascular disease. In disease, high density lipoprotein (HDL) particles lose their anti-inflammatory and antioxidant properties, becoming dysfunctional. The purpose of this study was to test the hypothesis that alterations in the HDL proteomic profile are associated with subclinical atherosclerosis and HDL dysfunction in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and type 1 diabetes. Targeted proteomics was used to quantify the relative abundance of 18 proteins in HDL from SLE patients with and without atherosclerotic plaque detectable by carotid ultrasound. Changes in the proteomic profile were compared against the in vitro ability of HDL to protect against lipid oxidation. The same proteins were quantified in HDL from patients with type 1 diabetes with or without coronary artery calcification as determined by computed tomography. In each population, paraoxonase-3 (PON3), a potent antioxidant protein, was depleted from the HDL of patients with subclinical atherosclerosis. PON3 expression in HDL was positively correlated with HDL antioxidant function. These results suggest that PON3 may be an important protein in preventing atherosclerosis and highlights the importance of antioxidant proteins in the prevention of atherosclerosis in vivo.

Background 25-Hydroxyvitamin D [25(OH)D] may be a poor marker of vitamin D status as it reflects differences in vitamin D binding protein (VDBP) between individuals. The vitamin D metabolite ratio [VMR, ratio of 24,25(OH)2D3 to 25(OH)D3] is a marker of vitamin D status that has been hypothesized to be independent of variability in VDBP. This hypothesis has not been directly evaluated. Methods We measured 25(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3, and VDBP in 377 community-dwelling older adults that participated in the Health Aging and Body Composition Study. 24,25(OH)2D3 and 25(OH)D3 were used to calculate the VMR. We used linear regression to assess the relationship between VDBP with the VMR, 24,25(OH)2D3, 25(OH)D3, and 1,25(OH)2D3. Results Participants had mean age 75 ± 3 years, 52% were female, 40% were black, and 24% had chronic kidney disease. VDBP concentrations were associated with sex, serum albumin, and VDBP phenotype in multivariable models. In fully adjusted models, each 1% higher VDBP was associated with a 0.92%[95% CI(0.37,1.49%)], 0.76% (0.39, 1.13%), and 0.57% (0.29, 0.85%), higher 24,25(OH)2D3, 25(OH)D3, and 1,25(OH)2D3. The VMR was independent of VDBP concentration, [0.16%(-0.11, 0.44) higher VMR per 1% higher VDBP, P = .25]. Conclusions The VMR was independent of VDBP concentration, whereas VDBP was strongly directly associated with the individual vitamin D metabolite concentrations. Prior studies evaluating only 25(OH)D3 may have been confounded by absence of data on VDBP status. The VMR may serve as an important biomarker of vitamin D status and clinical outcomes that can be utilized in populations with a large spectrum of VDBP concentrations.