Variant mapping using mass spectrometry-based proteotyping as a diagnostic tool in von Willebrand disease.

BACKGROUND: von Willebrand disease (VWD) is the most common inherited bleeding disorder, characterized by either partial or complete von Willebrand factor (VWF) deficiency or by the occurrence of VWF proteoforms of altered functionality. The gene encoding VWF is highly polymorphic, giving rise to a variety of proteoforms with varying plasma concentrations and clinical significance. OBJECTIVES: To address this complexity, we translated genomic variation in VWF to corresponding VWF proteoforms circulating in blood. METHODS: VWF was characterized in VWD patients (n = 64) participating in the Willebrand in the Netherlands study by conventional laboratory testing, DNA sequencing and complementary discovery, and targeted mass spectrometry-based plasma proteomic strategies. RESULTS: Unbiased plasma profiling combined with immune enrichment of VWF verified VWF and its binding partner factor VIII as key determinants of VWD and revealed a remarkable heterogeneity in VWF amino acid sequence coverage among patients. Subsequent VWF proteotyping enabled identification of both polymorphisms (eg, p.Thr789Ala, p.Gln852Arg, and p.Thr1381Ala), as well as pathogenic variants (n = 16) along with their corresponding canonical sequences. Targeted proteomics using stable isotope-labeled peptides confirmed unbiased proteotyping for 5 selected variants and suggested differential proteoform quantities in plasma. The variant-to-wild-type peptide ratio was determined in 6 type 2B patients heterozygous for p.Arg1306Trp, confirming the relatively low proteoform concentration of the pathogenic variant. The elevated VWF propeptide/VWF ratio indicated increased clearance of specific VWF proteoforms. CONCLUSION: This study highlights how VWF proteotyping from plasma could be the first step to bridge the gap between genotyping and functional testing in VWD.

Urinary Kidney Injury Biomarkers Are Associated with Ischemia-Reperfusion Injury Severity in Kidney Allograft Recipients.

BACKGROUND: We explored the potential of emerging and conventional urinary kidney injury biomarkers in recipients of living donor (LD) or donation after circulatory death (DCD) kidney transplantation, patients with chronic kidney disease (CKD), and individuals from the general population. METHODS: Urine samples from kidney allograft recipients with mild (LD; n = 199) or severe (DCD; n = 71) ischemia-reperfusion injury (IRI) were analyzed for neutrophil gelatinase-associated lipocalin (NGAL), insulin-like growth factor-binding protein 7 (IGFBP7), tissue inhibitor of metalloproteinases 2 (TIMP2), kidney injury molecule-1 (KIM-1), chemokine C-X-C motif (CXCL9), solute carrier family 22 member 2 (SLC22A2), nephrin, and uromodulin (UMOD) by quantitative multiplex LC-MS/MS analysis. The fold-change in biomarker levels was determined in mild and severe IRI and in patients with CKD stage 1-2 (n = 127) or stage ≥3 (n = 132) in comparison to the general population (n = 1438). Relationships between the biomarkers and total protein, ß2-microglobulin (B2M), creatinine, and osmolality were assessed. RESULTS: NGAL, IGFBP7, TIMP2, KIM-1, CXCL9, and UMOD were quantifiable, whereas nephrin and SLC22A2 were below the limit of detection. Kidney injury biomarkers were increased up to 6.2-fold in allograft recipients with mild IRI and 8.3-fold in recipients with severe IRI, compared to the reference population, with the strongest response observed for NGAL and B2M. In CKD stage 1-2, B2M, NGAL, IGFBP7, TIMP2, KIM-1, UMOD, and CXCL9 were not altered, but in individuals with CKD stage ≥3, B2M, NGAL, and KIM-1 were increased up to 1.3-fold. IGFBP7, TIMP2, NGAL, and CXCL9 were strongly correlated (all r ≥ 0.8); correlations with B2M and TP were smaller (all r ≤ 0.6). CONCLUSIONS: IRI, but not stable CKD, was associated with increased urinary levels of kidney injury biomarkers determined by LC-MS/MS. Absolute and multiplexed protein quantitation by LC-MS/MS is an effective strategy for biomarker panel evaluation for translation toward the clinical laboratory.

Reference intervals of urinary kidney injury biomarkers for middle-aged men and women determined by quantitative protein mass spectrometry.

BACKGROUND AND AIMS: There is an ongoing need to recognize early kidney injury and its progression in structural chronic pathologies. The proteins neutrophil-gelatinase-associated lipocalin (NGAL), insulin-like growth factor-binding protein 7 (IGFBP7), tissue inhibitor of metalloproteinases 2 (TIMP2), kidney injury molecule-1 (KIM-1), C-X-C motif chemokine 9 (CXCL9), transforming growth factor-beta 1 (TGF-ß1), solute carrier family 22 member 2 (SLC22A2), nephrin, cubilin, and uromodulin (UMOD) have been proposed as early kidney injury biomarkers. To guide clinical interpretation, their urinary concentrations should be accompanied by reference intervals, which we here establish in a representative Dutch middle-aged population. MATERIALS AND METHODS: The 24 h urine samples from 1443 Caucasian middle-aged men and women were analyzed for the biomarkers by quantitative LC-MS/MS. Biomarker excretion per 24 h were calculated, and urine creatinine and osmolality were measured for dilution normalization. This population was characterized by demographic and anthropometric parameters, comorbid conditions, and conventional kidney function measures. RESULTS: NGAL, IGFBP7, TIMP2, KIM-1, and UMOD could be quantified in this population, whereas nephrin, SLC22A2, and CXCL9 were below their detection limits. Urine creatinine and osmolality were correlated to urine volume (r = -0.71; -0.74) and to IGFBP7 (r = 0.73; 0.71) and TIMP2 (r = 0.71; 0.69). Crude and normalized biomarker concentrations were affected by sex, but not by age, body mass index, smoking, kidney function, or common comorbid conditions. The reference intervals (men; women) were 18-108; 21-131 pmol IGFBP7/mmol creatinine, 1-63; 4-224 pmol NGAL/mmol creatinine, 7-48; 7-59 pmol TIMP2/mmol creatinine, <1-9; <1-12 pmol KIM-1/mmol creatinine, and 0.1-1.2; 0.1-1.7 mg UMOD/mmol creatinine. CONCLUSION: We present dilution-normalized and sex-stratified urinary reference intervals of kidney injury biomarkers in a middle-aged Caucasian population.

Longitudinal nonlinear mixed effects modeling of EGFR mutations in ctDNA as predictor of disease progression in treatment of EGFR-mutant non-small cell lung cancer.

Correlations between increasing concentrations of circulating tumor DNA (ctDNA) in plasma and disease progression have been shown. A nonlinear mixed effects model to describe the dynamics of epidermal growth factor receptor (EGFR) ctDNA data from patients with non-small cell lung cancer (NSCLC) combined with a parametric survival model were developed to evaluate the ability of these modeling techniques to describe ctDNA data. Repeated ctDNA measurements on L858R, exon19del, and T790M mutants were available from 54 patients with EGFR mutated NSCLC treated with erlotinib or gefitinib. Different dynamic models were tested to describe the longitudinal ctDNA concentrations of the driver and resistance mutations. Subsequently, a parametric time-to-event model for progression-free survival (PFS) was developed. Predicted L858R, exon19del, and T790M concentrations were used to evaluate their value as predictor for disease progression. The ctDNA dynamics were best described by a model consisting of a zero-order increase and first-order elimination (19.7/day, 95% confidence interval [CI] 14.9-23.6/day) of ctDNA concentrations. In addition, time-dependent development of resistance (5.0 × 10-4 , 95% CI 2.0 × 10-4 -7.0 × 10-4 /day) was included in the final model. Relative change in L858R and exon19del concentrations from baseline was identified as most significant predictor of disease progression (p = 0.001). The dynamic model for L858R, exon19del, and T790M concentrations in ctDNA and time-to-event model adequately described the observed concentrations and PFS data in our clinical cohort. In addition, it was shown that nonlinear mixed effects modeling is a valuable method for the analysis of longitudinal and heterogeneous biomarker datasets obtained from clinical practice.

Multiplex LC-MS/MS Testing for Early Detection of Kidney Injury: A Next-Generation Alternative to Conventional Immunoassays?

BACKGROUND: LC-MS/MS has enabled the translation of many novel biomarkers to the clinical laboratory, but its potential for measurement of urinary proteins is still unexplored. In this study we examined the correlation and agreement between immunoassay and LC-MS/MS in the quantitation of kidney injury biomarkers and evaluated the application of technical LC-MS/MS meta-data assessment to ensure test result validity. METHODS: NGAL, IGFBP7, TIMP2, and KIM-1 were quantified in 345 urine samples with one multiplex lab-developed test that combines immunocapture with mass spectrometry read-out and 4 singleplex sandwich-type immunoassays. Assay performance and imprecision were monitored by 2 urine-based quality controls. Ion ratios, signal intensity, and retention time were monitored over all study samples. RESULTS: The LC-MS/MS retention time drift was ≤1.2%, ion ratios were within 20% of the target values at concentrations of >100 pmol/L, and peptides originating from the same protein were in agreement (slopes between 1.03 and 1.41). The interassay CV was between 9.3% and 19.1% for LC-MS/MS analysis and between 4.2% and 10.9% for immunoassay. Direct LC-MS/MS analysis was correlated with immunoassay in the quantitation of NGAL (r = 0.93; range: 0.01-37 nmol/L), IGFBP7 (r = 0.80; range: 0.01-2.6 nmol/L), TIMP2 (r = 0.85; range: 0.01-6.3 nmol/L), and KIM-1 (r = 0.70; range 0.01-0.4 nmol/L), but the analytical methodologies differed in measurands and calibration strategies. CONCLUSIONS: LC-MS/MS is explored as a next-generation technology for multiplex urinary protein measurement. It has great potential to overcome nonselectivity and lack of standardization because of its capability of directly measuring well-defined molecular proteins.

Development and Provisional Validation of a Multiplex LC-MRM-MS Test for Timely Kidney Injury Detection in Urine.

Kidney injury is a complication frequently encountered in hospitalized patients. Early detection of kidney injury prior to loss of renal function is an unmet clinical need that should be targeted by a protein-based biomarker panel. In this study, we aim to quantitate urinary kidney injury biomarkers at the picomolar to nanomolar level by liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode (LC-MRM-MS). Proteins were immunocaptured from urinary samples, denatured, reduced, alkylated, and digested into peptides before LC-MRM-MS analysis. Stable-isotope-labeled peptides functioned as internal standards, and biomarker concentrations were attained by an external calibration strategy. The method was evaluated for selectivity, carryover, matrix effects, linearity, and imprecision. The LC-MRM-MS method enabled the quantitation of KIM-1, NGAL, TIMP2, IGFBP7, CXCL9, nephrin, and SLC22A2 and the detection of TGF-ß1, cubilin, and uromodulin. Two to three peptides were included per protein, and three transitions were monitored per peptide for analytical selectivity. The analytical carryover was <1%, and minimal urine matrix effects were observed by combining immunocapture and targeted LC-MRM-MS analysis. The average total CV of all quantifier peptides was 26%. The linear measurement range was determined per measurand and found to be 0.05-30 nmol/L. The targeted MS-based method enables the multiplex quantitation of low-abundance urinary kidney injury biomarkers for future clinical evaluation.

Kidney Injury Biomarkers in an Academic Hospital Setting: Where Are We Now?

Acute kidney injury (AKI) is a frequent complication in hospitalised patients and is diagnosed by urinary output and serum creatinine. Serum creatinine is an indirect marker for renal glomerular filtration, but lacks specificity for damage to kidney tissue and the relatively late response to injury precludes early recognition of AKI. Timely diagnosis of kidney injury using biomarkers that provide information about the aetiology of kidney injury is an unmet clinical need. To overcome the suboptimal performance of serum creatinine, injury biomarkers have been proposed that predict AKI in diverse clinical settings. The clinical performance of these markers is considered moderate due to the lack of specificity for kidney tissue or the underlying injury mechanisms, poor test specificity and confounding by interventions or comorbidities. Hence, it is not unequivocally beneficial to implement current kidney injury biomarkers in the clinical laboratory for diagnostic purposes. In this article we review biomarkers that might fulfil AKI-related unmet clinical needs in the academic hospital setting.

A binding kinetics study of human adenosine A3 receptor agonists.

The human adenosine A3 (hA3) receptor has been suggested as a viable drug target in inflammatory diseases and in cancer. So far, a number of selective hA3 receptor agonists (e.g. IB-MECA and 2-Cl-IB-MECA) inducing anti-inflammatory or anticancer effects are under clinical investigation. Drug-target binding kinetics is increasingly recognized as another pharmacological parameter, next to affinity, for compound triage in the early phases of drug discovery. However, such a kinetics-driven analysis has not yet been performed for the hA3 receptor. In this study, we first validated a competition association assay for adenosine A3 receptor agonists to determine the target interaction kinetics. Affinities and Kinetic Rate Index (KRI) values of 11 ribofurano and 10 methanocarba nucleosides were determined in radioligand binding assays. Afterwards, 15 analogues were further selected (KRI <0.70 or KRI >1.35) for full kinetics characterization. The structure-kinetics relationships (SKR) were derived and longer residence times were associated with methanocarba and enlarged adenine N6 and C2 substitutions. In addition, from a kon-koff-KD kinetic map we divided the agonists into three subgroups. A residence time "cliff" was observed, which might be relevant to (N)-methanocarba derivatives' rigid C2-arylalkynyl substitutions. Our findings provide substantial evidence that, next to affinity, additional knowledge of binding kinetics is useful for developing and selecting new hA3R agonists in the early phase of the drug discovery process.

Structure-Affinity Relationships and Structure-Kinetics Relationships of Pyrido[2,1-f]purine-2,4-dione Derivatives as Human Adenosine A3 Receptor Antagonists.

We expanded on a series of pyrido[2,1-f]purine-2,4-dione derivatives as human adenosine A3 receptor (hA3R) antagonists to determine their kinetic profiles and affinities. Many compounds showed high affinities and a diverse range of kinetic profiles. We found hA3R antagonists with very short residence time (RT) at the receptor (2.2 min for 5) and much longer RTs (e.g., 376 min for 27 or 391 min for 31). Two representative antagonists (5 and 27) were tested in [35S]GTPγS binding assays, and their RTs appeared correlated to their (in)surmountable antagonism. From a kon-koff-KD kinetic map, we divided the antagonists into three subgroups, providing a possible direction for the further development of hA3R antagonists. Additionally, we performed a computational modeling study that sheds light on the crucial receptor interactions, dictating the compounds' binding kinetics. Knowledge of target binding kinetics appears useful for developing and triaging new hA3R antagonists in the early phase of drug discovery.