Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application

Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in ‘functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP

Blood amyloid and tau biomarkers as predictors of cerebrospinal fluid profiles

Blood biomarkers represent a major advance for improving the management, diagnosis, and monitoring of Alzheimer's disease (AD). However, their context of use in relation to routine cerebrospinal fluid (CSF) analysis for the quantification of amyloid peptides and tau proteins remains to be determined. We studied in two independent cohorts, the performance of blood biomarkers in detecting "nonpathological" (A−/T−/N−), amyloid (A+) or neurodegenerative (T+ /N+) CSF profiles. Plasma Aβ/Aβ ratio and phosphorylated tau (p-tau(181)) were independent and complementary predictors of the different CSF profile and in particular of the nonpathological (A−/T−/N−) profile with a sensitivity and specificity close to 85%. These performances and the corresponding biomarker thresholds were significantly different from those related to AD detection. The use of blood biomarkers to identify patients who may benefit from secondary CSF testing represents an attractive stratification strategy in the clinical management of patients visiting memory clinics. This could reduce the need for lumbar puncture and foreshadow the use of blood testing on larger populations. The online version contains supplementary material available at 10.1007/s00702-022-02474-9

Clinical perspectives of dried blood spot protein quantification using mass spectrometry methods

International audienceAlthough dried blood spot (DBS) sampling methods have been used since the 1960s, they have recently attracted renewed interest because of the development of new clinical applications. In addition to their other advantages, DBS methods can now be used to quantify many blood proteins using the latest highly sensitive and robust, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approaches such as multiple reaction monitoring. The DBS blood sampling approach could provide a useful alternative means of conducting blood sampling for routine clinical purposes and patients' follow-up. In this review, we examine the current use of DBS for LC-MS/MS protein quantification in clinical settings and discuss potential clinical applications

Comparison of Hydrophobic, Lipophilic and Immunodepletion Pre- Fractionation Methods for Label-Free LC-MS/MS Identification of Biomarkers in Human Cerebrospinal Fluid

International audienceBackground: Proteomics analysis of human cerebrospinal fluid (CSF) is a major tool for identifying novel biomarkers for neurological diseases. However, the complexity and wide dynamic range of CSF represent a major challenge for detecting specific low-abundance biomarkers. One way to overcome this problem is to rely on different pre-fractionation techniques. However, the most relevant technique remains to be determined. Methods: This study compared three different well-known pre-fractionation methods: immuno-depletion of major proteins (Seppro® IgY14), hydrophobic solid phase extraction (Oasis® HLB), and lipophilic sorbent concentration (Liposorb™). Unfractionated and pre-fractionated CSF was digested with trypsin and analyzed by RP-LC-MS/MS with an OrbitrapTM mass spectrometer. We documented the number of peptides detected and sets of proteins identified. Experiments were repeated to minimize pre-analytical and analytical variability.Results: Compared to unfractionated CSF, the OASIS® HLB fractionated CSF method showed a significant 28% increase in the total number of proteins identified, while the Liposorb™ capture resulted in a significant 46% decrease. Interestingly, results based on the number of peptides detected were different. We also evaluated the capacity of these pre-fractionation methods to detect different proteins in terms of their molecular weight, isoelectrophoretic point (IEP) or nature. Each of these pre-fractionation methods identified a specific subset of proteins, when compared to unfractionated CSF, and/or other methods. This was particularly obvious for the lipophilic sorbent, which allowed the detection of many lipoproteins.Conclusion: Direct analysis of digested CSF led to the identification of several proteins despite matrix complexity. As expected, single pre-fractionation methods that can be included in simple and cost-effective workflows, yielded significant differences in terms of number, or range of proteins identified. This suggests that a single pre-fractionation method cannot cover the full range of protein species present in a complex sampl

Nano-flow vs standard-flow: Which is the more suitable LC/MS method for quantifying hepcidin-25 in human serum in routine clinical settings?

International audienc

The calcium-dependent protein kinase CPK7 acts on root hydraulic conductivity

International audienceThe hydraulic conductivity of plant roots (Lpr ) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post-translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 (PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lpr of knockout Arabidopsis plants for four Ca(2+) -dependent protein kinases. cpk7 plants showed a 30% increase in Lpr because of a higher aquaporin activity. A quantitative proteomic analysis of wild-type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lpr of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7-dependent stability of specific membrane proteins

From radioimmunoassay to mass spectrometry: a new method to quantify orexin-A (hypocretin-1) in cerebrospinal fluid

International audienceI125 radioimmunoassay (RIA) is currently the standard technique for quantifying cerebrospinal fluid (CSF) orexin-A/hypocretin-1, a biomarker used to diagnose narcolepsy type 1. However, orexin-A RIA is liable to undergo cross-reactions with matrix constituents generating interference, high variability between batches, low precision and accuracy, and requires special radioactivity precautions. Here we developed the first quantitative mass spectrometry assay of orexin-A based on a multiple reaction monitoring (MRM) approach. This method was tested in keeping with the Clinical and Laboratory Standards Institute (CLSI) guidelines and its clinical relevance was confirmed by comparing patients with narcolepsy type 1 versus patients with other neurological conditions. The results obtained using MRM and RIA methods were highly correlated, and Bland–Altman analysis established their interchangeability. However, the MRM values had a wider distribution and were 2.5 time lower than the RIA findings. In conclusion, this method of assay provides a useful alternative to RIA to quantify orexin-A, and may well replace it not only in narcolepsy type 1, but also in the increasing number of pathologies in which the quantification of this analyte is relevant

Quantitative proteomics unravels novel post-translational regulatory mechanisms of plant aquaporins

International audienc

Quantitative proteomics unravels novel post-translational regulatory mechanisms of plant aquaporins

absen

β-Synuclein as a candidate blood biomarker for synaptic degeneration in Alzheimer’s disease

Abstract Synaptic degeneration is an early event closely associated with the course of Alzheimer’s disease (AD). The identification of synaptic blood biomarkers is, therefore, of great interest and clinical relevance. The levels of most synaptic proteins are increased in the cerebrospinal fluid (CSF) of patients with AD, but their detection in blood is hitherto either unavailable or not very informative. This paradigm is related to their low concentration, their peripheral origin, or the presence of highly abundant blood proteins that hinder detection. In recent years, significant progress has been made in detecting the presynaptic protein β-synuclein. This mini-review summarizes the results that highlight the role of β-synuclein as a candidate blood marker for synaptic degeneration in AD