In recent years, quantitative proteomic research attracts great attention because of the urgent needs in biological and clinical research, such as biomarker discovery and verification. Currently, mass spectrometry (MS) based bottom up strategy has become the method of choice for proteomic quantification. In this strategy, the amount of proteins is determined by quantifying the corresponding proteolytic peptides of the proteins, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed a new type of immobilized trypsin using hairy noncross-linked polymer chains hybrid magnetic nanoparticle as the matrix aiming at ultra fast, highly efficient proteomic digestion and facile (18)O labeling for absolution protein quantification. The hybrid nanoparticle is synthesized by in situ growth of hairy polymer chains from the magnetic nanoparticle surface using surface initiated atom transfer radical polymerization technique. The flexible noncross-linked polymer chains not only provide large amount of binding sites but also work as scaffolds to support three-dimensional trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin. For complex proteomic samples, obviously increased digestion efficiency and completeness was demonstrated by 27.2% and 40.8% increase in the number of identified proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from Thermoanaerobacter tengcongensis protein extracts using (18)O labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin for high throughput proteome quantification.
osteosarcoma (oS) is the most common bone cancer in children and young adults. Solid tumors are characterized by intratumoral hypoxia, and hypoxic cells are associated with the transformation to aggressive phenotype and metastasis. the proteome needed to support an aggressive osteosarcoma cell phenotype remains largely undefined. To link metastatic propensity to a hypoxia-induced proteotype, we compared the protein profiles of two isogenic canine OS cell lines, POS (low metastatic) and HMpoS (highly metastatic), under normoxia and hypoxia. Label-free shotgun proteomics was applied to comprehensively characterize the hypoxia-responsive proteome profiles in the OS cell phenotypes. Hypothesis-driven parallel reaction monitoring was used to validate the differential proteins observed in the shotgun data and to monitor proteins of which we expected to exhibit hypoxia responsiveness, but which were absent in the label-free shotgun data. We established a "distance" score (|z HMPOS − z POS |), and "sensitivity" score (|z Hypoxia − z Normoxia ) to quantitatively evaluate the proteome shifts exhibited by oS cells in response to hypoxia. evaluation of the sensitivity scores for the proteome shifts observed and principal component analysis of the hypoxia-responsive proteins indicated that both cell types acquire a proteome that supports a Warburg phenotype with enhanced cell migration and proliferation characteristics. Cell migration and glucose uptake assays combined with protein function inhibitor studies provided further support that hypoxia-driven adaption of pathways associated with glycolytic metabolism, collagen biosynthesis and remodeling, redox regulation and immunomodulatory proteins typify a proteotype associated with an aggressive cancer cell phenotype. Our findings further suggest that proteins involved in collagen remodeling and immune editing may warrant further evaluation as potential targets for anti-metastatic treatment strategies in osteosarcoma.
In an age of whole-genome analysis, the mass spectrometry-based bottom-up strategy is now considered to be the most powerful method for in-depth proteomics analysis. As part of this strategy, highly efficient and complete proteolytic digestion of proteins into peptides is crucial for successful proteome profiling with deep coverage. To achieve this goal, prolonged digestion time and the use of multiple proteases have been adopted. The long digestion time required and tedious sample treatment steps severely limit the sample processing throughput. Though utilization of immobilized protease greatly reduces the digestion time, highly efficient proteolysis of extremely complex proteomic samples remains a challenging task. Here, we propose a dual matrix-based complementary digestion method using two types of immobilized trypsin with opposite matrix hydrophobicity prepared by attaching trypsin on hydrophobic or hydrophilic polymer-brush-modified nanoparticles. The polymer brushes on the nanoparticles serve as three-dimensional supports for a large amount of trypsin immobilization and lead to ultrafast and highly efficient protein digestion. More importantly, the two types of immobilized trypsin show high complementarity in protein digestion with only ∼60% overlap in peptide identification for yeast and membrane protein of mouse liver. Complementary digestion by applying these two types of immobilized trypsin together leads to obviously enhanced protein and peptide identification. Furthermore, the dual matrix-based complementary digestion shows particular advantage in the digestion of membrane proteins, as twice the number of identified peptides is obtained compared with solution digestion using free proteases, demonstrating its potential as a promising alternative to promote proteomics analysis with higher protein sequence coverage.
Here we developed a facile method using the polycyclic aromatic hydrocarbon ethidium bromide (EB) for glycan derivatization, to enhance the ionization efficiency and detection sensitivity of glycans in mass spectrometry (MS). The cationic nature of EB also notably improved identification of neutral and acidic glycans of real samples, as well as their structural analysis.
Coronary microvascular dysfunction (MVD) is a syndrome of abnormal regulation of vascular tone, particularly during increased metabolic demand. While there are several risk factors for MVD, some of which are similar to those for coronary artery disease (CAD), the cause of MVD is not understood. We hypothesized that MVD in symptomatic non-elderly subjects would be characterized by specific lipidomic profiles. Subjects (n = 20) aged 35–60 years and referred for computed tomography coronary angiography (CTA) for chest pain but who lacked obstructive CAD (>50% stenosis), underwent quantitative regadenoson stress-rest myocardial contrast echocardiography (MCE) perfusion imaging for MVD assessment. The presence of MVD defined by kinetic analysis of MCE data was correlated with lipidomic profiles in plasma measured by liquid chromatography and high-resolution mass spectrometry. Nine of twenty subjects had evidence of MVD, defined by reduced hyperemic perfusion versus other subjects (beta-value 1.62 ± 0.44 vs. 2.63 ± 0.99 s−1, p = 0.009). Neither the presence of high-risk but non-obstructive CAD on CTA, nor CAD risk factors were different for those with versus without MVD. Lipidomic analysis revealed that patients with MVD had lower concentrations of long-carbon chain triacylglycerols and diacylglycerols, and higher concentrations of short-chain triacylglycerols. The diacylglycerol containing stearic and linoleic acid classified all participants correctly. We conclude that specific lipidomic plasma profiles occur in MVD involving saturated long-chain fatty acid-containing acylglycerols that are distinctly different from those in non-obstructive CAD. These patterns could be used to better characterize the pathobiology and potential treatments for this condition.
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