Microchip gel electrophoretic analysis of perchloric acid‐soluble serum proteins in systemic inflammatory disorders

Abstract

Perchloric acid (PCA) precipitation is a well‐known method for the separation of heavily glycosylated proteins and for reducing the masking effect of major serum proteins. The aim of this study is to characterize PCA‐soluble serum proteins in healthy individuals and in patients with systemic inflammatory diseases, such as Crohn's disease and sepsis. A PCA precipitation protocol was prepared and adapted to the analytical methods. After PCA treatment of the serum, the soluble proteins in the supernatant were analyzed by SDS‐PAGE and by microchip gel electrophoresis (MGE). Characteristic changes of the electrophoretic patterns of the PCA‐soluble fractions were observed. Four characteristic bands (at ∼11, ∼65, ∼85, and ∼120 kDa) with varying intensity were detected by MGE. The proportion of the ∼65, ∼85, and ∼120 kDa bands were significantly higher in systemic inflammatory conditions than in healthy individuals ( p < 0.001), and characteristic patterns were observed in patients with acute inflammation. The marked differences in the acid‐soluble protein patterns, which were observed in patients with ongoing systemic inflammation, might be a good indicator of inflammation. The MGE analysis is a fast screening and quantification method for the detection of characteristic changes among acid‐soluble serum proteins.

Plasma Proteomic Analysis Distinguishes Severity Outcomes of Human Ebola Virus Disease

“…We developed a cost-effective HTP LC-MS-based plasma proteomics pipeline based on perchloric acid protein precipitation, which robustly removes the abundant plasma proteins and allows the detection of less abundant plasma proteins. While several proteomic studies have employed perchloric acid precipitation as a semiselective depletion/enrichment strategy ( 31 – 33 ) on small numbers of samples, we have advanced this protocol into a robust HTP-compatible large-scale plasma proteomic method. The main advantages of this depletion method include (i) the extreme precipitation conditions, which make the protocol impervious to variations in the starting conditions of the samples; (ii) the low cost per sample; and (iii) its compatibility with 96-well plates and liquid-handling robots.…”

Efficacy of lyophilised bacteria-rich faecal sediment and supernatant with reduced bacterial count for treating patients with Clostridioides difficile Infection – A novel method for capsule faecal microbiota transfer

“…The collected stool samples were analysed with microchip gel electrophoresis (protocol below from Makszin et al. (2018) ).…”

Determination of Glycoproteins by Microchip Electrophoresis Using Os(VI)-Based Selective Electrochemical Tag

“…Transferrin (Tf) and α-1-acid glycoprotein (AGP) are the main glycoproteins present in human serum, which have been used as biomarkers and therapeutic targets in clinical diagnosis. , These glycoproteins could provide clinically relevant information for early recognition and monitoring of systemic inflammatory disorders such as Crohn’s disease and sepsis because their concentration can increase up to 2–3 times in some of these diseases. − …”