Quantification of Proteins in Blood by Absorptive Microtiter Plate-Based Affinity Purification Coupled to Liquid Chromatography-Mass Spectrometry.

Liquid chromatography (LC) coupled to mass spectrometry (MS) is increasingly used for quantification of proteins in blood. This development is prompted by ongoing improvements in detection sensitivities of LC-MS instruments and corresponding sample preparation workflows. The combination of immunoaffinity enrichment and targeted LC-MS detection is a notable analytical platform in this regard as it allows for the quantification of low abundance proteins in biological matrices like plasma and serum. Here, we describe such hybrid methods which are based on the enrichment of proteins with antibodies or affimers coupled to adsorptive microtiter plates, the proteolytic digestion of enriched proteins to release protein-specific peptides, and the detection of these peptides by microflow LC coupled to selected reaction monitoring MS.

Assessing the Potential of Untargeted SWATH Mass Spectrometry-Based Metabolomics to Differentiate Closely Related Exposures in Observational Studies.

Mass spectrometry (MS) is increasingly used in clinical studies to obtain molecular evidence of chemical exposures, such as tobacco smoke, alcohol, and drugs. This evidence can help verify clinical data retrieved through anamnesis or questionnaires and may provide insights into unreported exposures, for example those classified as the same despite small but possibly relevant chemical differences or due to contaminants in reported exposure compounds. Here, we aimed to explore the potential of untargeted SWATH metabolomics to differentiate such closely related exposures. This data-independent acquisition MS-based profiling technique was applied to urine samples of 316 liver and 570 kidney transplant recipients from the TransplantLines Biobank and Cohort Study (NCT03272841), where we focused on the immunosuppressive drug mycophenolate, which is either supplied as a morpholino-ester prodrug or as an enteric-coated product, the illicit drug cocaine, which is usually supplied as an adulterated product, and the proton pump inhibitors omeprazole and esomeprazole. Based on these examples, we found that untargeted SWATH metabolomics has considerable potential to identify different (unreported) exposure or co-exposure metabolites and may determine variations in their abundances. We also found that these signals alone may sometimes be unable to distinguish closely related exposures, and enhancement of differentiation, for example by integration with pharmacogenomics data, is needed.

Soluble receptor for advanced glycation end products (sRAGE) as a biomarker of COPD.

BACKGROUND: Soluble receptor for advanced glycation end products (sRAGE) is a proposed emphysema and airflow obstruction biomarker; however, previous publications have shown inconsistent associations and only one study has investigate the association between sRAGE and emphysema. No cohorts have examined the association between sRAGE and progressive decline of lung function. There have also been no evaluation of assay compatibility, receiver operating characteristics, and little examination of the effect of genetic variability in non-white population. This manuscript addresses these deficiencies and introduces novel data from Pittsburgh COPD SCCOR and as well as novel work on airflow obstruction. A meta-analysis is used to quantify sRAGE associations with clinical phenotypes. METHODS: sRAGE was measured in four independent longitudinal cohorts on different analytic assays: COPDGene (n = 1443); SPIROMICS (n = 1623); ECLIPSE (n = 2349); Pittsburgh COPD SCCOR (n = 399). We constructed adjusted linear mixed models to determine associations of sRAGE with baseline and follow up forced expiratory volume at one second (FEV1) and emphysema by quantitative high-resolution CT lung density at the 15th percentile (adjusted for total lung capacity). RESULTS: Lower plasma or serum sRAGE values were associated with a COPD diagnosis (P < 0.001), reduced FEV1 (P < 0.001), and emphysema severity (P < 0.001). In an inverse-variance weighted meta-analysis, one SD lower log10-transformed sRAGE was associated with 105 ± 22 mL lower FEV1 and 4.14 ± 0.55 g/L lower adjusted lung density. After adjusting for covariates, lower sRAGE at baseline was associated with greater FEV1 decline and emphysema progression only in the ECLIPSE cohort. Non-Hispanic white subjects carrying the rs2070600 minor allele (A) and non-Hispanic African Americans carrying the rs2071288 minor allele (A) had lower sRAGE measurements compare to those with the major allele, but their emphysema-sRAGE regression slopes were similar. CONCLUSIONS: Lower blood sRAGE is associated with more severe airflow obstruction and emphysema, but associations with progression are inconsistent in the cohorts analyzed. In these cohorts, genotype influenced sRAGE measurements and strengthened variance modelling. Thus, genotype should be included in sRAGE evaluations.

SWATH data independent acquisition mass spectrometry for screening of xenobiotics in biological fluids: Opportunities and challenges for data processing.

SWATH data independent acquisition (DIA) mass spectrometry (MS) has become an established technique in MS-based 'omics' research and is increasingly used for the screening of xenobiotics (e.g. drugs, drug metabolites, pesticides, toxicants). Such xenobiotic screening methods are mostly applied for tentative compound identification purposes based on spectral library searching, while additional data processing techniques are scarcely used thereby leaving the full potential of these methods often unused. Here we present an analytical workflow for screening xenobiotics in human samples using SWATH/MS based on which we highlight opportunities for unlocking unused potential of these methods. The workflow was applied to urine samples from subjects who tested positive for THC and/or cocaine during roadside drug testing with the goal of confirming the positive roadside drug tests and identifying compounds that relate to illicit drug use (e.g. cutting agents, tobacco components) or associate with corresponding lifestyle choices (e.g. nasal decongestants, painkillers). These goals could only be reached by complementing spectral library search procedures with additional multivariate data analyses due to inherent incompleteness of the spectral library that was employed. Such incompleteness represents a common challenge for applications where limited or no metadata is available for study samples, for example in toxicology, doping control in sports, and workplace or roadside drug testing. It furthermore sets the stage for employing additional data processing techniques as is outlined in the presented work.

Affimers as an Alternative to Antibodies in an Affinity LC-MS Assay for Quantification of the Soluble Receptor of Advanced Glycation End-Products (sRAGE) in Human Serum.

Antibodies are indispensable tools in biomedical research, but their size, complexity, and sometimes lack of reproducibility created a need for the development of alternative binders to overcome these limitations. Affimers are a novel class of affinity binders based on a structurally robust protease inhibitor scaffold (i.e., Cystatin A), which are selected by phage display and produced in a rapid and simple E. coli protein expression system. These binders have a defined amino acid sequence with defined binding regions and are versatile, thereby allowing for easy engineering. Here we present an affimer-based liquid chromatography-mass spectrometry (LC-MS) method for quantification of the soluble Receptor of Advanced Glycation End-products (sRAGE), a promising biomarker for chronic obstructive pulmonary disease. The method was validated according to European Medicines Agency and U.S. Food and Drug Administration guidelines and enabled quantitation of serum sRAGE between 0.2 and 10 ng/mL. Comparison between the affimer-based method and a previously developed, validated antibody-based method showed good correlation ( R2 = 0.88) and indicated that 25% lower sRAGE levels are reported by the affimer-based assay. In conclusion, we show the first-time application of affimers in a quantitative LC-MS method, which supports the potential of affimers as robust alternatives to antibodies.

Assessment of Sample Preparation Bias in Mass Spectrometry-Based Proteomics.

For mass spectrometry-based proteomics, the selected sample preparation strategy is a key determinant for information that will be obtained. However, the corresponding selection is often not based on a fit-for-purpose evaluation. Here we report a comparison of in-gel (IGD), in-solution (ISD), on-filter (OFD), and on-pellet digestion (OPD) workflows on the basis of targeted (QconCAT-multiple reaction monitoring (MRM) method for mitochondrial proteins) and discovery proteomics (data-dependent acquisition, DDA) analyses using three different human head and neck tissues (i.e., nasal polyps, parotid gland, and palatine tonsils). Our study reveals differences between the sample preparation methods, for example, with respect to protein and peptide losses, quantification variability, protocol-induced methionine oxidation, and asparagine/glutamine deamidation as well as identification of cysteine-containing peptides. However, none of the methods performed best for all types of tissues, which argues against the existence of a universal sample preparation method for proteome analysis.

Single-nucleotide polymorphism in the 5-α-reductase gene (SRD5A2) is associated with increased prevalence of metabolic syndrome in chemotherapy-treated testicular cancer survivors.

PURPOSE: Chemotherapy-treated testicular cancer survivors are at risk for development of the metabolic syndrome, especially in case of decreased androgen levels. Polymorphisms in the gene encoding steroid 5-α-reductase type II (SRD5A2) are involved in altered androgen metabolism. We investigated whether single-nucleotide polymorphisms (SNPs) rs523349 (V89L) and rs9282858 (A49T) in SRD5A2 are associated with cardiometabolic status in testicular cancer survivors. METHODS: In 173 chemotherapy-treated testicular cancer survivors, hormone levels and cardiometabolic status were evaluated cross-sectionally (median 5 years [range 3-20] after chemotherapy) and correlated with SNPs in SRD5A2. RESULTS: The metabolic syndrome was more prevalent in survivors who were homozygous or heterozygous variant for SRD5A2 rs523349 compared to wild type (33% versus 19%, P = 0.032). In particular, patients with lower testosterone levels (<15 nmol/l) and a variant genotype showed a high prevalence of the metabolic syndrome (66.7%). Mean intima-media thickness of the carotid artery and urinary albumin excretion, both markers of vascular damage, were higher in the group of survivors homozygous or heterozygous variant for rs523349 (0.62 versus 0.57 mm, P = 0.026; 5.6 versus 3.1 mg/24 h, P = 0.017, respectively). No association was found between cardiometabolic status and SNP rs9282858 in SRD5A2. CONCLUSION: Metabolic syndrome develops more frequently in testicular cancer survivors homozygous or heterozygous variant for SNP rs523349 in SRD5A2. Altered androgen sensitivity appears to be involved in the development of adverse metabolic and vascular changes in testicular cancer survivors and is a target for intervention.