Establishment of measurement traceability for peptide and protein quantification through rigorous purity assessment—a review

Abstract Current dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum apolipoproteins A-I, (a), B, C-I, C-II, C-III, and E may give better pathophysiological insight into the root causes of dyslipidemia. To facilitate the future adoption of clinical serum apolipoprotein (apo) profiling for precision medicine, strategies for accurate testing should be developed in advance. Recent discoveries in basic science and translational medicine set the stage for the IFCC Working Group on Apolipoproteins by Mass Spectrometry. Main drivers were the convergence of unmet clinical needs in cardiovascular disease (CVD) patients with enabling technology and metrology. First, the residual cardiovascular risk after accounting for established risk factors demonstrates that the current lipid panel is too limited to capture the full complexity of lipid metabolism in patients. Second, there is a need for accurate test results in highly polymorphic and atherogenic apolipoproteins such as apo(a). Third, sufficient robustness of mass spectrometry technology allows reproducible protein quantification at the molecular level. Fourth, several calibration hierarchies in the revised ISO 17511:2020 guideline facilitate metrological traceability of test results, the highest achievable standard being traceability to SI. This article outlines the conceptual approach aimed at achieving a novel, multiplexed Reference Measurement System (RMS) for seven apolipoproteins based on isotope dilution mass spectrometry and peptide-based calibration. This RMS should enable standardization of existing and emerging apolipoprotein assays to SI, within allowable limits of measurement uncertainty, through a sustainable network of Reference Laboratories.

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Kinetic mechanism of Na+-coupled aspartate transport catalyzed by GltTk

Communications Biology ◽

10.1038/s42003-021-02267-y ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Gianluca Trinco ◽

Valentina Arkhipova ◽

Alisa A. Garaeva ◽

Cedric A. J. Hutter ◽

Markus A. Seeger ◽

...

Keyword(s):

Kinetic Mechanism ◽

Protein Quantification ◽

Detergent Solution ◽

Sodium Ions ◽

Binding Studies ◽

Equilibrium Binding ◽

Uptake Rates ◽

Bona Fide ◽

Inside Out

AbstractIt is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified GltTk, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on GltPh using this equation allowed for determination of the turnover number (0.14 s−1), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented GltTk proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging.

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Rapid highly sensitive general protein quantification through on-chip chemiluminescence

Biomicrofluidics ◽

10.1063/5.0039872 ◽

2021 ◽

Vol 15 (2) ◽

pp. 024113

Author(s):

Hoi Kei Chiu ◽

Tadas Kartanas ◽

Kadi L. Saar ◽

Carina Mouritsen Luxhøj ◽

Sean Devenish ◽

...

Keyword(s):

Protein Quantification ◽

Highly Sensitive ◽

General Protein ◽

On Chip

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Advances in absolute protein quantification and quantitative protein mapping using ICP-MS

TrAC Trends in Analytical Chemistry ◽

10.1016/j.trac.2017.09.024 ◽

2018 ◽

Vol 104 ◽

pp. 148-159 ◽

Cited By ~ 13

Author(s):

Laura Cid-Barrio ◽

Francisco Calderón-Celis ◽

Patricia Abásolo-Linares ◽

M. Luisa Fernández-Sánchez ◽

José Manuel Costa-Fernández ◽

...

Keyword(s):

Protein Quantification ◽

Protein Mapping ◽

Icp Ms

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Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

Molecules ◽

10.3390/molecules26082206 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2206

Author(s):

Thai Pham ◽

Renjie Liao ◽

Joshua Labaer ◽

Jia Guo

Keyword(s):

High Performance ◽

Single Cells ◽

Protein Quantification ◽

Cellular Systems ◽

Protein Profiling ◽

High Signal ◽

Protein Targets ◽

Chemical Reagents ◽

Ffpe Tissues

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.

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Stroke promotes the development of brain atrophy and delayed cell death in hypertensive rats

Scientific Reports ◽

10.1038/s41598-020-75450-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Mohammed A. Sayed ◽

Wael Eldahshan ◽

Mahmoud Abdelbary ◽

Bindu Pillai ◽

Waleed Althomali ◽

...

Keyword(s):

Cell Death ◽

Fold Increase ◽

Magnetic Resonance Images ◽

Protein Quantification ◽

Motor Deficits ◽

Long Term Memory ◽

Delayed Presentation ◽

Hypertensive Rats ◽

Post Stroke ◽

Delayed Cell Death

AbstractPost-stroke cognitive impairment (PSCI) is a major source of disability, affecting up to two thirds of stroke survivors with no available therapeutic options. The condition remains understudied in preclinical models due to its delayed presentation. Although hypertension is a leading risk factor for dementia, how ischemic stroke contributes to this neurodegenerative condition is unknown. In this study, we used a model of hypertension to study the development of PSCI and its mechanisms. Spontaneously hypertensive rats (SHR) were compared to normotensive rats and were subjected to 1-h middle cerebral artery occlusion or sham surgery. Novel object recognition, passive avoidance test and Morris water maze were used to assess cognition. In addition, brain magnetic resonance images were obtained 12-weeks post-stroke and tissue was collected for immunohistochemistry and protein quantification. Stroked animals developed impairment in long-term memory at 4-weeks post-stroke despite recovery from motor deficits, with hypertensive animals showing some symptoms of anhedonia. Stroked SHRs displayed grey matter atrophy and had a two-fold increase in apoptosis in the ischemic borderzone and increased markers of inflammatory cell death and DNA damage at 12 weeks post-stroke. This indicates that preexisting hypertension exacerbates the development of secondary neurodegeneration after stroke beyond its acute effects on neurovascular injury.

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