Differential proteomic patterns of plasma extracellular vesicles show potential to discriminate ß-thalassemia subtypes.

The observed specificity of ß-thalassemia-subtype phenotypes makes new diagnostic strategies that complement current screening methods necessary to determine each subtype and facilitate therapeutic regimens for different patients. Here, we performed quantitative proteomics of plasma-derived extracellular vesicles (EVs) of ß-thalassemia major (TM) patients, ß-thalassemia intermedia (TI) patients, and healthy controls to explore subgroup characteristics and potential biomarkers. Plasma quantitative proteomics among the same cohorts were analyzed in parallel to compare the biomarker potential of both specimens. EV proteomics showed significantly more abnormalities in immunity and lipid metabolism in TI and TM, respectively. The differential proteomic patterns of EVs were consistent with but more striking than those of plasma. Notably, we also found EV proteins to have a superior performance for discriminating ß-thalassemia subtypes. These findings allowed us to propose a diagnostic model consisting of five proteins in EVs with subtyping potential, demonstrating the ability of plasma-derived EVs for the diagnosis of ß-thalassemia patients.

Comprehensive Evaluation of Different TiO2-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics.

Protein phosphorylation is an essential post-translational modification that regulates multiple cellular processes. Due to their low stoichiometry and ionization efficiency, it is critical to efficiently enrich phosphopeptides for phosphoproteomics. Several phosphopeptide enrichment methods have been reported; however, few studies have comprehensively compared different TiO2-based phosphopeptide enrichment methods using complex proteomic samples. Here, we compared four TiO2-based phosphopeptide enrichment methods that used four non-phosphopeptide excluders (glutamic acid, lactic acid, glycolic acid, and DHB). We found that these four TiO2-based phosphopeptide enrichment methods had different enrichment specificities and that phosphopeptides enriched by the four methods had different physicochemical characteristics. More importantly, we discovered that phosphopeptides had a higher deamidation ratio than peptides from cell lysate and that phosphopeptides enriched using the glutamic acid method had a higher deamidation ratio than the other three methods. We then compared two phosphopeptide fractionation methods: ammonia- or TEA-based high pH reversed-phase (HpH-RP). We found that fewer phosphopeptides, especially multi-phosphorylated peptides, were identified using the ammonia-based method than using the TEA-based method. Therefore, the TEA-based HpH-RP fractionation method performed better than the ammonia method. In conclusion, we comprehensively evaluated different TiO2-based phosphopeptide enrichment and fractionation methods, providing a basis for selecting the proper protocols for comprehensive phosphoproteomics.

Plasma proteome profiling combined with clinical and genetic features reveals the pathophysiological characteristics of ß-thalassemia.

The phenotype of ß-thalassemia underlies multigene interactions, making clinical stratification complicated. An increasing number of genetic modifiers affecting the disease severity have been identified, but are still unable to meet the demand of precision diagnosis. Here, we systematically conducted a comparative plasma proteomic profiling on patients with ß-thalassemia and healthy controls. Among 246 dysregulated proteins, 13 core protein signatures with excellent biomarker potential are proposed. The combination of proteome and patients' clinical data revealed patients with codons 41/42 -TTCT mutations have an elevated risk of higher iron burden, dysplasia, and osteoporosis than patients with other genotypes. Notably, 85 proteins correlating to fetal hemoglobin (Hb F) were identified, among which the abundance of 27 proteins may affect the transfusion burden in patients with ß-thalassemia. The current study thus provides protein signatures as potential diagnostic biomarkers or therapeutic clues for ß-thalassemia.

Separation and characterization of extracellular vesicles from human plasma by asymmetrical flow field-flow fractionation.

It is a big challenge to isolate extracellular vesicles (EVs) from human plasma because of the contamination from high abundant lipoproteins, such as high density lipoprotein (HDL) and low density lipoprotein particles (LDL). In this study, the parameters of asymmetrical flow field-flow fractionation (AF4) technology and sample preparation, including cross flow gradient, focusing time, ultrafiltration condition, sample amount and injection volume have been optimized and successfully utilized for the separation and characterization of EVs from human plasma. This study demonstrated that the great potential of AF4 in the separation of EVs from HDL and LDL in human plasma with high reproducibility and purity. This study indicated excessive focusing time in the AF4 separation and 100-300 kDa MWCO membrane based ultrafiltration in the pre-preparation will cause loss of EVs. A total of 1038 proteins have been identified in seven replicates of purified EVs from pooled human plasma sample. They are mainly enriched in extracellular exosomes, involved in extracellular matrix structural constituent, and associated with extracellular matrix-receptor interaction pathway. This study also indicated that human plasma contains more EVs than the paired serum at the same volume, and showed age- and gender-independent individual variability of the amount of EVs in human plasma. This study displayed that AF4 technique can serve as a powerful platform for the separation of EVs from human plasma, serum or human body fluids and this technology will promote the studies on EVs, such as proteomics, biomarker discovery and functions.