Label-free quantitative proteomic analysis of serum extracellular vesicles differentiating patients of alcoholic and nonalcoholic fatty liver diseases.

Alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are typically asymptomatic and slow-progressing but potentially fatal diseases that are common causes of liver cirrhosis and related complications. Exosomes are nano-sized extracellular vesicles that have been linked to various intercellular communication processes and can carry biological materials reflecting the state and severity of disease. In this study, shotgun proteomic analysis of the protein expression profiles of extracellular vesicles, including exosomes and microvesicles, enriched from human serum samples of 24 patients diagnosed with various fatty liver diseases was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS) followed by protein identification and label-free quantification using the MaxQuant platform. A total of 329 proteins, including 190 previously reported exosome-specific proteins, were identified from four types of liver disease, where significant differences in protein expression were found in apolipoproteins, immunoglobulins, and other previously reported markers of liver disease. Principal component analysis of 61 proteins identified from MaxQuant analysis of the LC-MS/MS data provided a confident differentiation between ALD and NAFLD. SIGNIFICANCE: The current investigation revealed the difference among various types of liver disease using LC-MS/MS of exosomes enriched from human serum samples of 24 patients where the most significantly up-regulation proteins were alpha-2-macroglobulin for alcoholic hepatitis and apolipoprotein C3 for nonalcoholic fatty liver disease.

MALDI-MS analysis of disaccharide isomers using graphene oxide as MALDI matrix.

Disaccharides are sugars composed of two monosaccharides joined by a glycosidic linkage. The specific properties of a disaccharide depend on the type of the glycosidic linkage and the identity of the two component monosaccharides. In this work, seven disaccharide isomers (gentiobiose, isomaltose, melibiose, lactose, maltose, cellobiose, and sucrose) were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using a graphene oxide matrix. Each disaccharide was identified by its unique cleavage pattern. To determine the feasibility of quantitative analyses based on specific fragment patterns, mixtures of sucrose with cellobiose or maltose were prepared at different ratios and analyzed by MALDI-MS, where a strong linear correlation was observed between the relative peak intensity of the sucrose fragment peak at m/z 185 and the amount of sucrose in the mixture. The calibration curve was successfully applied to obtain the relative amount of maltose and sucrose in four different honey samples.

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

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.

Effects of Matrices and Additives on Multiple Charge Formation of Proteins in MALDI-MS Analysis.

The sinapinic acid (SA) matrix has frequently been used for protein analysis in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). However, the SA matrix does not result in the formation of distinctive multiple protein charge states, whereas the 2-nitrophloroglucinol (2-NPG) matrix is capable of this. The formation of multiple charge states in the MALDI-MS analysis of proteins is advantageous in that it results in higher accuracy. In this study, the mass spectra of several common standard proteins, namely cytochrome c, myoglobin, bovine serum albumin (BSA), and immunoglobulin G (IgG), were compared using various matrices (2,5-dihydroxybenzoic acid, α-cyano-hydroxycinnamic acid, SA, and 2-NPG). Furthermore, the mass spectra of two large standard proteins (BSA and IgG) using various acid additives (H3PO4, HNO3, H2SO4, HCl, and trifluoroacetic acid) with the 2-NPG matrix were also compared. Among the different matrices, 2-NPG provided the broadest range of multiple protein charge states, while, among the different additives, the 2-NPG matrix in combination with HCl generated the broadest multiple charge states as well as the most intense protein peaks.

Effects of incubation temperature and acetonitrile amount on microwave-assisted tryptic digestion of proteins.

The effects of incubation temperature and acetonitrile (ACN) amount on microwave-assisted tryptic digestion of horse skeletal muscle myoglobin (MYG) and bovine serum albumin (BSA) were investigated. Microwave-assisted tryptic digestion was performed on BSA or MYG solutions containing different amounts (0, 10, and 20%) of ACN for different times (10, 20, 30, 40, and 50 min) at different temperatures (25, 37, and 55 °C). Conventional overnight tryptic digestion was also conducted with gentle mixing at 37 °C for 16 h. Digested samples were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. Similar sequence coverage (SC) values were obtained under most conditions except when the protein sample solutions were digested with 20% ACN at 55 °C, which provided the lowest SC for both MYG and BSA for all investigated digestion times. Considering the missed cleavage (MC) ratios for 50-min microwave-assisted digestion, the highest MC ratio, (i.e., lowest trypsin activity) was observed for the digestion condition of 20% ACN at 55 °C for both proteins, while the lowest MC ratio was observed with 0% ACN at 25 °C for MYG and with 0% ACN at 55 °C for BSA. Conventional overnight tryptic digestion at 37 °C provided more completely cleaved peptides than 50-min microwave-assisted tryptic digestion at the same temperature.