Quantitative Mass Spectrometry Evaluation of Human Retinol Binding Protein 4 and Related Variants

Background: Retinol Binding Protein 4 (RBP4) is an exciting new biomarker for the determination of insulin resistance and type 2 diabetes. It is known that circulating RBP4 resides in multiple variants which may provide enhanced clinical utility, but conventional immunoassay methods are blind to such differences. A Mass Spectrometric immunoassay (MSIA) technology that can quantitate total RBP4 as well as individual isoforms may provide an enhanced analysis for this biomarker. Methods: RBP4 was isolated and detected from 0.5 uL of human plasma using MSIA technology, for the simultaneous quantification and differentiation of endogenous human RBP4 and its variants. Results: The linear range of the assay was 7.81–500 ug/mL, and the limit of detection and limit of quantification were 3.36 ug/mL and 6.52 ug/mL, respectively. The intra-assay CVs were determined to be 5.1 % and the inter-assay CVs were 9.6%. The percent recovery of the RBP4-MSIA ranged from 95 – 105%. Method comparison of the RBP4 MSIA vs the Immun Diagnostik ELISA yielded a Passing & Bablok fit of MSIA = 1.056 ELISA – 3.09, while the Cusum linearity p-value was.0.1 and the mean bias determined by the Altman Bland test was 1.2%. Conclusion: The novel RBP4 MSIA provided a fast, accurate and precise quantitative protein measurement as compared to the standard commercially available ELISA. Moreover, this method also allowed for the detection of RBP4 variants that are present in each sample, which may in the future provide a new dimension in the clinical utility of this biomarker

Kлинички тек и иÑход кај пациенти Ñо рано дијагноÑтицирани конгенитални малформации на бубрезите и уринарниот тракт

Congenital anomalies of the kidney and urinary tract- CAKUT are common childhood pathology accounting for 20-30% of all perinatally detected congenital anomalies. The importance of CAKUT is the risk they represent to kidney function deterioration and end-stage renal disease development.The aim of this study was to analyze the clinical course and outcome in patients with early diagnosed CAKUT. The study was designed as retrospective observational study.  A total of 100 patients with early diagnosed CAKUT at University Children's Hospital, Skopje, were enrolled in the study. The results obtained in this study present the clinical course and outcome in the study group. Urinary tract  infections were seen in 30% of patients enrolled in this study; they were predominantly girls (65%), mostly  diagnosed with posterior urethral  valve, VUR (vesicouretral reflux) and UPJO (ureteropelviuc junction obstruction). Surgical treatment  was required in 28% of patients, and chronic renal failure was diagnosed in 4% of the total number of patients in this study.This study aims to contribute to understanding the early diagnosed CAKUT as well as to establishing a protocol for early detection and adequate treatment in order to prevent renal function deterioration. ÐšÐ¾Ð½Ð³ÐµÐ½Ð¸Ñ‚алните малформации на бубрезите и уринарниот тракт (congenital anomalies of the kidney and urinary tract- CAKUT) Ñе чеÑти во детÑката возраÑÑ‚ и Ñочинуваат помеѓу 20 и 30% од Ñите конге- нитални аномалии во перинаталниот период. Значењето на конгениталните аномалии на бубрезите и уринарниот тракт е ризикот што тие го ноÑат за влошување на бубрежната функција Ñо развој на прогреÑивна бубрежна болеÑÑ‚ и бубрежна инÑуфициенција. Целта на оваа Ñтудија беше да Ñе опи- ше клиничкиот тек и иÑходот кај пациентите Ñо рано дијагноÑтициран CAKUT. Студијата  Ð±ÐµÑˆÐµ ди- зајнирана како ретроÑпективна опÑеревациона Ñтудија. Таа опфати 100 иÑпитаници Ñо пренатално поÑтавено Ñомнение за конгенитална малформација на бубрезите и уринарниот тракт. Пациентите беа прегледани на УниверзитетÑката клиника за детÑки болеÑти, Скопје. Резултатите ги прикажуваат клиничкиот тек и иÑходот кај пациентите Ñо рана дијагноза на конгенитални аномалии на бубрезите и уринарниот тракт. Уринарни инфекции во тек на Ñледењето беа региÑтрирани кај 30% од пациентите, почеÑто кај девојчиња (65%), во поголем процент заÑтапени кај пациенти Ñо валвула на задна уретра, VUR (vesicouretral reflux) и UPJO (ureteropelviuc junction obstruction). Оперативен третман беше не- опходен кај 28% од пациентите, а хронична бубрежна болеÑÑ‚ беше дијагноÑтицирана кај 4%. Оваа Ñтудија има за цел да придонеÑе во разбирањето на карактериÑтиките на рано детектираните конге- нитални малформации и поÑтавување на Ñтратегија за рана поÑтнатална потврда на пренатално по- Ñтавеното Ñомнение за малформација и ран, оптимизиран третман, Ñо цел редукција или одложување на прогреÑија на детериорација на бубрежната функција и терминална бубрежна болеÑÑ‚

New Multiscale Characterization Methodology for Effective Determination of Isolation-Structure-Function Relationship of Extracellular Vesicles

Extracellular vesicles (EVs) have been lauded as next-generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass produce EVs, i.e., to isolate, purify, and characterize them effectively. Technical limitations in comprehensive characterization of EVs lead to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were likely to contribute to the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterization approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies

Targeted quantitative mass spectrometric immunoassay for human protein variants

Abstract Background Post-translational modifications and genetic variations give rise to protein variants that significantly increase the complexity of the human proteome. Modified proteins also play an important role in biological processes. While sandwich immunoassays are routinely used to determine protein concentrations, they are oblivious to protein variants that may serve as biomarkers with better sensitivity and specificity than their wild-type proteins. Mass spectrometry, coupled to immunoaffinity separations, can provide an efficient mean for simultaneous detection and quantification of protein variants. Results Presented here is a mass spectrometric immunoassay method for targeted quantitative proteomics analysis of protein modifications. Cystatin C, a cysteine proteinase inhibitor and a potential marker for several pathological processes, was used as a target analyte. An internal reference standard was incorporated into the assay, serving as a normalization point for cystatin C quantification. The precision, linearity, and recovery characteristics of the assay were established. The new assay was also benchmarked against existing cystatin C ELISA. In application, the assay was utilized to determine the individual concentration of several cystatin C variants across a cohort of samples, demonstrating the ability to fully quantify individual forms of post-translationally modified proteins. Conclusions The mass spectrometric immunoassays can find use in quantifying specific protein modifications, either as a part of a specific protein biomarker discovery/rediscovery effort to delineate the role of these variants in the onset of the disease, progression, and response to therapy, or in a more systematic study to delineate and understand human protein diversity.</p

Mass Spectrometric Immunoassays in Characterization of Clinically Significant Proteoforms

Proteins can exist as multiple proteoforms in vivo, as a result of alternative splicing and single-nucleotide polymorphisms (SNPs), as well as posttranslational processing. To address their clinical significance in a context of diagnostic information, proteoforms require a more in-depth analysis. Mass spectrometric immunoassays (MSIA) have been devised for studying structural diversity in human proteins. MSIA enables protein profiling in a simple and high-throughput manner, by combining the selectivity of targeted immunoassays, with the specificity of mass spectrometric detection. MSIA has been used for qualitative and quantitative analysis of single and multiple proteoforms, distinguishing between normal fluctuations and changes related to clinical conditions. This mini review offers an overview of the development and application of mass spectrometric immunoassays for clinical and population proteomics studies. Provided are examples of some recent developments, and also discussed are the trends and challenges in mass spectrometry-based immunoassays for the next-phase of clinical applications

Parallel Workflow for High-Throughput (.1,000 Samples/Day) Quantitative Analysis of Human Insulin-Like Growth Factor 1 Using Mass Spectrometric Immunoassay

Abstract Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories. Presented here is an MS-based quantitative IGF1 assay with performance rating of .1,000 samples/day, and a capability of quantifying IGF1 point mutations and posttranslational modifications. The throughput of the IGF1 mass spectrometric immunoassay (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a tip format, and high-throughput MALDI-TOF MS analysis. The Limit of Detection and Limit of Quantification of the resulting assay were 1.5 mg/L and 5 mg/L, respectively, with intra-and inter-assay precision CVs of less than 10%, and good linearity and recovery characteristics. The IGF1 MSIA was benchmarked against commercially available IGF1 ELISA via Bland-Altman method comparison test, resulting in a slight positive bias of 16%. The IGF1 MSIA was employed in an optimized parallel workflow utilizing two pipetting robots and MALDI-TOF-MS instruments synced into one-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data collection, and data processing. Using this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This rate of assaying is a significant improvement over existing MS-based IGF1 assays, and is on par with that of the enzyme-based immunoassays. Furthermore, a mutation was detected in ,1% of the samples (SNP: rs17884626, creating an ART substitution at position 67 of the IGF1), demonstrating the capability of IGF1 MSIA to detect point mutations and posttranslational modifications