Platelet Factor 4 as a Novel Exosome Marker in MALDI-MS Analysis of Exosomes from Human Serum

Abstract

Exosomes are nanosized vesicles commonly found in biological fluids as a result of a secretion process involving endosomes and multivesicular bodies. The isolation and analysis of exosomes can be useful for noninvasive clinical diagnosis of a variety of human diseases. We investigated the utility of analyzing exosomal proteins, using matrix-assisted laser desorption/ionization combined with Fourier-transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS), as a means of determining the presence of exosomes. MALDI-FTICR-MS analyses of exosomes enriched from human serum via centrifugation in a mass range of m/z 1000–20 000 yielded a distinctive protein around m/z 7766. The high mass accuracy and resolution of MALDI-FTICR-MS allowed for reliable comparisons against a protein database, through which the protein was identified as platelet factor 4 (PLF4), whose singly charged protein peak has an elemental composition of C341H577N96O101S4 +, with a theoretical most abundant isotopic peak at m/z 7765.194 and a theoretical average peak at m/z 7766. The MALDI-TOF MS analysis of exosomes from the serum of 27 patients with different states of liver diseases provided the most abundant PLF4 peak for each mass spectrum, along with several additional minor peaks. In conclusion, MALDI-MS is suitable as an alternative exosome detection method, serving as a valuable confirmation tool, greatly decreasing the time and workload associated with exosome identification.

Analysis of the regenerative capacity of human serum exosomes after a simple multistep separation from lipoproteins

“…One of the most prominent was properdin, which is an immune‐response‐linked protein. Interestingly, we also detected platelet factor 4 which has been recently identified as a novel exosome marker for human exosome serum (Nguyen et al, 2019).…”

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

“…These proteins are generally used as marker proteins for exosomes. Moreover, a recently study on the exosomes enriched from human serum identified with high confidence by MALDI-MS analysis the platelet factor 4 as a novel exosome marker that can be used as an alternative tool for confirming the exosome presence [ 149 ].…”

Extracellular vesicles as a promising biomarker resource in liquid biopsy for cancer

“…Several housekeeping genes or reference genes have been identified for different native tissues and body fluids, and stable endogenous RNAs have been proposed as internal controls; however, a consensus has not been reached [152] . In 2002, Vandesompele et al [153] demonstrated that the common RT-qPCR procedure of using only one control gene induced relatively large errors. In this study, they claimed that ideal single internal control genes do not exist and recommended the use of at least three adequate control genes for calculating a normalization factor [153] .…”

“…In 2002, Vandesompele et al [153] demonstrated that the common RT-qPCR procedure of using only one control gene induced relatively large errors. In this study, they claimed that ideal single internal control genes do not exist and recommended the use of at least three adequate control genes for calculating a normalization factor [153] . This issue with normalizers in gene expression analyses continues to this day, and the same issue is found with protein analyses.…”