Asiatic acid (AA; 2α,3β,23-trihydroxyurs-12-ene-28-oic acid) is widely used for medicinal purposes in many Asian countries due to its various bioactivities. A series of AA derivatives has been synthesized in attempts to improve its therapeutic potencies. Herein we investigated the anti-tumor activities of N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester (AA-PMe), a novel AA derivative. AA-PMe exhibited a stronger anti-cancer activity than its parent compound AA. AA-PMe inhibited the proliferation of SGC7901 and HGC27 human gastric cancer cells in a dose-dependent manner but had no significant toxicity in human gastric mucosa epithelial cells (GES-1). AA-PMe induced cell cycle arrest in G0/G1 phase and blocked G1-S transition, which correlated well with marked decreases in levels of cyclin D1, cyclin-dependent kinase CKD4, and phosphorylated retinoblastoma protein, and increase in cyclin-dependent kinase inhibitor P15. Further, AA-PMe induced apoptosis of human gastric cancer cells by affecting Bcl-2, Bax, c-Myc, and caspase-3. Moreover, AA-PMe suppressed the migration and invasion of human gastric cancer cells (SGC7901 and HGC27) cells by downregulating the expression of MMP-2 and MMP-9. Overall, this study investigated the potential anti-cancer activities of AA-PMe including inducing apoptosis and suppressing proliferation, migration and invasion of gastric cancer cells, as well as the underlying mechanisms, suggesting that AA-PMe is a promising anti-cancer drug candidate in gastric cancer therapy.
Asiatic acid derivatives; gastric cancer cells; anti-tumor effect; cytotoxicity; apoptosis; cell cycle arrest; migration; invasion; mobility
Tissue proteomics is increasingly recognized for its role in biomarker discovery and disease mechanism investigation. However, protein solubility remains a significant challenge in mass spectrometry (MS)-based tissue proteomics. Conventional surfactants such as sodium dodecyl sulfate (SDS), the preferred surfactant for protein solubilization, are not compatible with MS. Herein, we have screened a library of surfactant-like compounds and discovered an MS-compatible degradable surfactant (MaSDeS) for tissue proteomics that solubilizes all categories of proteins with performance comparable to SDS. The use of MaSDeS in the tissue extraction significantly improves the total number of protein identifications from commonly used tissues, including tissue from the heart, liver, and lung. Notably, MaSDeS significantly enriches membrane proteins, which are often under-represented in proteomics studies. The acid degradable nature of MaSDeS makes it amenable for high-throughput mass spectrometry-based proteomics. In addition, the thermostability of MaSDeS allows for its use in experiments requiring high temperature to facilitate protein extraction and solubilization. Furthermore, we have shown that MaSDeS outperforms the other MS-compatible surfactants in terms of overall protein solubility and the total number of identified proteins in tissue proteomics. Thus, the use of MaSDeS will greatly advance tissue proteomics and realize its potential in basic biomedical and clinical research. MaSDeS could be utilized in a variety of proteomics studies as well as general biochemical and biological experiments that employ surfactants for protein solubilization.
Surfactant; Proteomics; Membrane Proteins; Mass Spectrometry; Tissue
The specific role of AMPKα1 or AMPKα2 in mediating cardiomyocyte contractile function remains elusive. The present study investigated how AMPK activation modulates the contractility of isolated cardiomyocytes.
Mechanical properties and intracellular Ca2+ properties were measured in isolated cardiomyocytes. The stress signaling was evaluated using western blot and immunoprecipitation analysis.
AMPK activator, A-769662 induced maximal velocity of shortening (+dL/dt) and relengthening (−dL/dt), peak height and peak shortening (PS) amplitude in both WT and AMPKα2 KO cardiomyocytes, but did not affect time-to-90% relengthening (TR90). AMPK KD cardiomyocytes demonstrated contractile dysfunction compared with cardiomyocytes from WT and AMPKα2 KO hearts. However, the rise of intracellular Ca2+ levels as well as intracellular ATP levels has no significant difference among WT, AMPKα2 KO and AMPK KD groups with and without the presence of A-769662. Besides, WT, AMPKα2 KO and AMPK KD group displayed a phosphorylated AMPK and downstream acetyl-CoA carboxylase (ACC) phosphorylation. Interestingly, A-769662 also triggered troponin I (cTnI) phosphorylation at Ser149 site which is related to contractility of cardiomyocytes. Furthermore, the immunoprecipitation analysis revealed that AMPKα1 of cardiomyocytes was phosphorylated by A-769662.
This is the first study illustrating that activation of AMPK plays a significant role in mediating the contractile function of cardiomyocytes using transgenic animal models. AMPK activator facilitates the contractility of cardiomyocytes via activating AMPKα1 catalytic subunit. The phosphorylation of cTnI by AMPK could be a factor attributing to the regulation of contractility of cardiomyocytes.
Contractile function; AMPK; Troponin I
Analysis of protein phosphorylation remains a significant challenge due to the low abundance of phosphoproteins and the low stoichiometry of phosphorylation, which requires effective enrichment of phosphoproteins. Here we have developed superparamagnetic nanoparticles (NPs) whose surface is functionalized by multivalent ligand molecules that specifically bind to the phosphate groups on any phosphoproteins. These NPs enrich phosphoproteins from complex cell and tissue lysates with high specificity as confirmed by SDS-PAGE analysis with a phosphoprotein-specific stain and mass spectrometry analysis of the enriched phosphoproteins. This method enables universal and effective capture, enrichment, and detection of intact phosphoproteins towards a comprehensive analysis of the phosphoproteome.
Ginsenoside Rg1 (Rg1) has anti-aging and anti-neurodegenerative effects. However, the mechanisms underlying these actions remain unclear. The aim of the present study was to determine whether Rg1 affects hippocampal survival and neurite outgrowth in vitro after exposure to amyloid-beta peptide fragment 25–35 (Aβ25–35), and to explore whether the extracellular signal-regulated kinase (ERK) and Akt signaling pathways are involved in these biological processes. We cultured hippocampal neurons from newborn rats for 24 hours, then added Rg1 to the medium for another 24 hours, with or without pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) family or Akt signaling pathways for a further 24 hours. We then immunostained the neurons for growth associated protein-43, and measured neurite length. In a separate experiment, we exposed cultured hippocampal neurons to Aβ25–35 for 30 minutes, before adding Rg1 for 48 hours, with or without Akt or MAPK inhibitors, and assessed neuronal survival using Hoechst 33258 staining, and phosphorylation of ERK1/2 and Akt by western blot analysis. Rg1 induced neurite outgrowth, and this effect was blocked by API-2 (Akt inhibitor) and PD98059 (MAPK/ERK kinase inhibitor), but not by SP600125 or SB203580 (inhibitors of c-Jun N-terminal kinase and p38 MAPK, respectively). Consistent with this effect, Rg1 upregulated the phosphorylation of Akt and ERK1/2; these effects were reversed by API-2 and PD98059, respectively. In addition, Rg1 significantly reversed Aβ25–35-induced apoptosis; this effect was blocked by API-2 and PD98059, but not by SP600125 or SB203580. Finally, Rg1 significantly reversed the Aβ25–35-induced decrease in Akt and ERK1/2 phosphorylation, but API-2 prevented this reversal. Our results indicate that Rg1 enhances neurite outgrowth and protects against Aβ25–35-induced damage, and that its mechanism may involve the activation of Akt and ERK1/2 signaling.
nerve regeneration; ginsenoside Rg1; neurite outgrowth; Aβ25–35; hippocampal neurons; Akt; MAPK; apoptosis; growth associated protein-43; Hoechst 33258 staining; PD98059; API-2; neural regeneration
Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function.
Biomedicine; Cardiac regeneration; Heart disease; Label-free quantification; Large mammalian models; Stem cells
Human induced pluripotent stem cells (hiPSCs) hold promise for myocardial repair following injury, but preclinical studies in large animal models are required to determine optimal cell preparation and delivery strategies to maximize functional benefits and to evaluate safety. Here, we utilized a porcine model of acute myocardial infarction (MI) to investigate the functional impact of intramyocardial transplantation of hiPSC-derived cardiomyocytes, endothelial cells, and smooth muscle cells, in combination with a 3D fibrin patch loaded with insulin growth factor (IGF)-encapsulated microspheres. hiPSC-derived cardiomyocytes integrated into host myocardium and generated organized sarcomeric structures, and endothelial and smooth muscle cells contributed to host vasculature. Tri-lineage cell transplantation significantly improved left ventricular function, myocardial metabolism, and arteriole density, while reducing infarct size, ventricular wall stress and apoptosis without inducing ventricular arrhythmias. These findings in a large animal MI model highlight the potential of utilizing hiPSC-derived cells for cardiac repair.
human induced pluripotent stem cells; cell therapy; metabolism; myocardial infarction; heart
ubiquitylation, one of the most prevalent post-translational
modifications in eukaryotes, is involved in regulating nearly every
cellular signaling pathway. The vast functional range of ubiquitylation
has largely been attributed to the formation of a diverse array of
polymeric ubiquitin (polyUb) chains. Methods that enable the characterization
of these diverse chains are necessary to fully understand how differences
in structure relate to function. Here, we describe a method for the
detection of enzymatically derived branched polyUb conjugates in which
a single Ub subunit is modified by two Ub molecules at distinct lysine
residues. Using a middle-down mass spectrometry approach in which
restricted trypsin-mediated digestion is coupled with mass spectrometric
analysis, we characterize the polyUb chains produced by bacterial
effector E3 ligases NleL (non-Lee-encoded effector ligase from enterohemorrhagic Escherichia coli O157:H7) and IpaH9.8 (from Shigella
flexneri). Because Ub is largely intact after minimal trypsinolysis,
multiple modifications on a single Ub moiety can be detected. Analysis
of NleL- and IpaH9.8-derived polyUb chains reveals branch points are
present in approximately 10% of the overall chain population. When
unanchored, well-defined polyUb chains are added to reaction mixtures
containing NleL, longer chains are more likely to be modified internally,
forming branch points rather than extending from the end of the chain.
These results suggest that middle-down mass spectrometry can be used
to assess the extent to which branched polyUb chains are formed by
various enzymatic systems and potentially evaluate the presence of
these atypical conjugates in cell and tissue extracts.
of the challenges in proteomics is the proteome’s complexity,
which necessitates the fractionation of proteins prior to the mass
spectrometry (MS) analysis. Despite recent advances in top-down proteomics,
separation of intact proteins remains challenging. Hydrophobic interaction
chromatography (HIC) appears to be a promising method that provides
high-resolution separation of intact proteins, but unfortunately the
salts conventionally used for HIC are incompatible with MS. In this
study, we have identified ammonium tartrate as a MS-compatible salt
for HIC with comparable separation performance as the conventionally
used ammonium sulfate. Furthermore, we found that the selectivity
obtained with ammonium tartrate in the HIC mobile phases is orthogonal
to that of reverse phase chromatography (RPC). By coupling HIC and
RPC as a novel two-dimensional chromatographic method, we have achieved
effective high-resolution intact protein separation as demonstrated
with standard protein mixtures and a complex cell lysate. Subsequently,
the separated intact proteins were identified by high-resolution top-down
MS. For the first time, these results have shown the high potential
of HIC as a high-resolution protein separation method for top-down
The pathophysiology of heart failure (HF) is diverse, owing to multiple etiologies and aberrations in a number of cellular processes. Therefore, it is essential to understand how defects in the molecular pathways that mediate cellular responses to internal and external stressors function as a system to drive the HF phenotype. Mass spectrometry (MS)-based proteomics strategies have great potential for advancing our understanding of disease mechanisms at the systems level because proteins are the effector molecules for all cell functions and, thus, are directly responsible for determining cell phenotype. Two MS-based proteomics strategies exist: peptide-based bottom-up and protein-based top-down proteomics—each with its own unique strengths and weaknesses for interrogating the proteome. In this review, we will discuss the advantages and disadvantages of bottom-up and top-down MS for protein identification, quantification, and the analysis of post-translational modifications, as well as highlight how both of these strategies have contributed to our understanding of the molecular and cellular mechanisms underlying HF. Additionally, the challenges associated with both proteomics approaches will be discussed and insights will be offered regarding the future of MS-based proteomics in HF research.
Heart failure; proteomics; mass spectrometry; post-translational modifications; systems biology
To determine serum cytokine profiles in Graves' disease (GD) patients with or without active and inactive thyroid associated ophthalmopathy (TAO), we recruited 65 subjects: 10 GD only (without TAO), 25 GD + active TAO, 20 GD + TAO, and 10 healthy controls. Liquid chip assay was used to measure serum Th1/Th2/Th17 cytokines including IFN-γ (interferon-gamma), TNF-α (tumor necrosis factor-alpha), IL-1α (interleukin-1 alpha), IL-1Ra (IL-1 receptor antagonist), IL-2, IL-4, IL-6, and IL-17 and two chemokines: RANTES (regulated upon activation, normal T cell expressed and secreted) and IP-10 (IFN-γ-induced protein 10). Serum levels of TSH (thyroid stimulating hormone) receptor autoantibodies (TRAb) were measured using an enzyme linked immunosorbent assay. Compared with healthy controls, TAO patients showed significantly elevated serum levels of IFN-γ, TNF-α, IL-1α, IL-4, IL-6, IL-17, and IP-10. Comparing active and inactive TAO, serum Th1 cytokines IFN-γ and TNF-α were elevated in active TAO, while serum Th2 cytokine IL-4 was elevated in inactive TAO. Serum Th17 cytokine IL-17 was elevated in GD but reduced in both active and inactive TAO. A positive correlation was found between TRAb and IFN-γ, TNF-α, IL-1α, IL-2, IL-4, and IL-6. Taken together, serum Th1/Th2/Th17 cytokines and chemokines reflect TAO disease activity and may be implicated in TAO pathogenesis.
Proteomics is essential for deciphering how molecules interact as a system and for understanding the functions of cellular systems in human disease; however, the unique characteristics of the human proteome, which include a high dynamic range of protein expression and extreme complexity due to a plethora of post-translational modifications (PTMs) and sequence variations, make such analyses challenging. An emerging “top-down” mass spectrometry (MS)-based proteomics approach, which provides a “bird’s eye” view of all proteoforms, has unique advantages for the assessment of PTMs and sequence variations. Recently, a number of studies have showcased the potential of top-down proteomics for unraveling of disease mechanisms and discovery of new biomarkers. Nevertheless, the top-down approach still faces significant challenges in terms of protein solubility, separation, and the detection of large intact proteins, as well as the under-developed data analysis tools. Consequently, new technological developments are urgently needed to advance the field of top-down proteomics. Herein, we intend to provide an overview of the recent applications of top-down proteomics in biomedical research. Moreover, we will outline the challenges and opportunities facing top-down proteomics strategies aimed at understanding and diagnosing human diseases.
Proteomics; mass spectrometry; human disease; post-translational modifications; systems biology
Protein ubiquitylation, one of the most prevalent post-translational modifications in eukaryotes, is involved in regulating nearly every cellular signaling pathway. The vast functional range of ubiquitylation has largely been attributed to the formation of a diverse array of polymeric ubiquitin (polyUb) chains. Methods that enable the characterization of these diverse chains are necessary to fully understand how differences in structure relate to function. Here, we describe a method for the detection of enzymatically derived branched polyUb conjugates in which a single Ub subunit is modified by two Ub molecules at distinct lysine residues. Using a middle-down mass spectrometry approach in which restricted trypsin-mediated digestion is coupled with mass spectrometric analysis, we characterize the polyUb chains produced by bacterial effector E3 ligases NleL (non-Lee-encoded effector ligase from enterohemorrhagic Escherichia coli O157:H7) and IpaH9.8 (from Shigella flexneri). Because Ub is largely intact after minimal trypsinolysis, multiple modifications on a single Ub moiety can be detected. Analysis of NleL- and IpaH9.8-derived polyUb chains reveals branch points are present in approximately 10% of the overall chain population. When unanchored, well-defined polyUb chains are added to reaction mixtures containing NleL, longer chains are more likely to be modified internally, forming branch points rather than extending from the end of the chain. These results suggest that middle-down mass spectrometry can be used to assess the extent to which branched polyUb chains are formed by various enzymatic systems and potentially evaluate the presence of these atypical conjugates in cell and tissue extracts.
Chemical methods for modifying proteins can enable studies aimed at uncovering biochemical function. Herein, we describe the use of thiol–ene coupling (TEC) chemistry to report on the function of branched (also referred to as forked) ubiquitin trimers. We show how site-specific isopeptide (Nε-Gly-l-homothiaLys) bonds are forged between two molecules of Ub, demonstrating the power of TEC in protein conjugation. Moreover, we demonstrate that the Nε-Gly-l-homothiaLys isopeptide bond is processed to a similar extent by deubiquitinases (DUBs) as that of a native Nε-Gly-l-Lys isopeptide bond, thereby establishing the utility of TEC in the generation of Ub-Ub linkages. TEC is then applied to the synthesis of branched Ub trimers. Interrogation of these branched derivatives with DUBs reveals that the relative orientation of the two Ub units has a dramatic impact on how they are hydrolyzed. In particular, cleavage of K48C-linkages is suppressed when the central Ub unit is also conjugated through K6C, whereas cleavage proceeds normally when the central unit is conjugated through either K11C or K63C. The results of this work presage a role for branched polymeric Ub chains in regulating linkage-selective interactions.
The rapid advancements in mass spectrometry (MS) instrumentation, particularly in Fourier transform (FT) MS, have made the acquisition of high-resolution and high-accuracy mass measurements routine. However, the software tools for the interpretation of high-resolution MS data are underdeveloped. Although several algorithms for the automatic processing of high-resolution MS data are available, there is still an urgent need for a user-friendly interface with functions that allow users to visualize and validate the computational output. Therefore, we have developed MASH Suite, a user-friendly and versatile software interface for processing high-resolution MS data. MASH Suite contains a wide range of features that allow users to easily navigate through data analysis, visualize complex high-resolution MS data and manually validate automatically processed results. Furthermore, it provides easy, fast and reliable interpretation of top-down, middle-down and bottom-up MS data. MASH Suite is convenient, easily operated and freely available. It can greatly facilitate the comprehensive interpretation and validation of high-resolution MS data with high accuracy and reliability.
Top-down Mass Spectrometry; Tandem Mass Spectrometry; Electron Capture Dissociation; Data processing; Bioinformatics
The formation of disulfide bonds between cysteine residues is crucial for the stabilization of native protein structures and, thus, determination of disulfide linkages is an important facet of protein structural characterization. Nonetheless, the identification of disulfide bond linkages remains a significant analytical challenge, particularly in large proteins with complex disulfide patterns. Herein, we have developed a new liquid chromatography mass spectrometric (LC/MS) strategy for rapid screening of disulfides in an intact protein mixture after a straightforward reduction step with tris(2-carboxyethyl)phosphine. LC/MS analysis of reduced and non-reduced protein mixtures quickly revealed disulfide-containing proteins owing to a 2 Da mass increase per disulfide reduction and, subsequently, the total number of disulfide bonds in the intact proteins could be determined. We have demonstrated the effectiveness of this method in a protein mixture composed of both disulfide-containing and disulfide-free proteins. Our method is simple (no need for proteolytic digestion, alkylation, or the removal of reducing agents prior to MS analysis), high throughput (fast on-line LC/MS analysis) and reliable (no S-S scrambling), underscoring its potential as a rapid disulfide screening method for proteomics applications.
Disulfide; Intact Proteins; Proteomics; Liquid Chromatography; TCEP; Mass Spectrometry
Docetaxel is an adjuvant chemotherapy drug widely used to treat multiple solid tumors; however, its toxicity and side effects limit its clinical efficacy. Herein, docetaxel-loaded solid lipid nanoparticles (DSNs) were developed to reduce systemic toxicity of docetaxel while still keeping its anticancer activity. To evaluate its anticancer activity and toxicity, and to understand the molecular mechanisms of DSNs, different cellular, molecular, and whole genome transcription analysis approaches were utilized. The DSNs showed lower cytotoxicity compared with the commercial formulation of docetaxel (Taxotere®) and induced more apoptosis at 24 hours after treatment in vitro. DSNs can cause the treated cancer cells to arrest in the G2/M phase in a dose-dependent manner similar to Taxotere. They can also suppress tumor growth very effectively in a mice model with human xenograft breast cancer. Systemic analysis of gene expression profiles by microarray and subsequent verification experiments suggested that both DSNs and Taxotere regulate gene expression and gene function, including DNA replication, DNA damage response, cell proliferation, apoptosis, and cell cycle regulation. Some of these genes expressed differentially at the protein level although their messenger RNA expression level was similar under Taxotere and DSN treatment. Moreover, DSNs improved the main side effect of Taxotere by greatly lowering myelosuppression toxicity to bone marrow cells from mice. Taken together, these results expound the antitumor efficacy and the potential working mechanisms of DSNs in its anticancer activity and toxicity, which provide a theoretical foundation to develop and apply a more efficient docetaxel formulation to treat cancer patients.
docetaxel; docetaxel-loaded solid lipid nanoparticles; breast cancer; toxicity
Seasonal estrus is a critical limiting factor of animal fecundity, and it involves changes in both ovarian biology and hormone secretion in different seasons. Previous studies indicate that two classes of small RNAs (miRNAs and piRNAs) play important regulatory roles in ovarian biology. To understand the roles of small RNA-mediated post-transcriptional regulation in ovine seasonal estrus, the variation in expression patterns of ovarian small RNAs during anestrus and the breeding season were analyzed using Solexa sequencing technology. In addition, reproductive hormone levels were determined during ovine anestrus and the breeding season.
A total of 483 miRNAs (including 97 known, 369 conserved and 17 predicated novel miRNAs), which belong to 183 different miRNA families, were identified in ovaries of Tan sheep and Small Tail Han (STH) sheep. Compared with the three stages of the breeding season, 25 shared significantly differentially expressed (including 19 up- and six down-regulated) miRNAs were identified in ovine anestrus. KEGG Pathway analysis revealed that the target genes for some of the differentially expressed miRNAs were involved in reproductive hormone related pathways (e.g. steroid biosynthesis, androgen and estrogen metabolism and GnRH signaling pathway) as well as follicular/luteal development related pathways. Moreover, the expression of the differentially expressed miRNAs and most of their target genes were negatively correlated in the above pathways. Furthermore, the levels of estrogen, progesterone and LH in ovine anestrus were significantly lower than those in the breeding season. Combining the results of pathway enrichment analysis, expression of target genes and hormone measurement, we suggest that these differentially expressed miRNAs in anestrus might participate in attenuation of ovarian activity by regulating the above pathways. Besides miRNAs, a large and unexpectedly diverse set of piRNAs were also identified.
The miRNA profiles of ovine ovaries in anestrus were presented for the first time. The identification and characterization of miRNAs that are differentially expressed between ovine anestrus and the breeding season will help understanding of the role of miRNAs in the regulation of seasonal estrus, and provides candidates for determining miRNAs which could be potentially used to regulate ovine seasonal estrus.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-899) contains supplementary material, which is available to authorized users.
Sheep; Seasonal estrus; Anestrus; Ovary; miRNA; piRNA
Top-down mass spectrometry (MS)-based proteomics has gained a solid growth over the past few years but still faces significant challenges in the liquid chromatographic separation of intact proteins. In top-down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. Size exclusion chromatography (SEC) is a favored liquid chromatography method for size-based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein we reported the use of ultra-high pressure (UHP) SEC for rapid and high-resolution separation of intact proteins for top-down proteomics. Fast separation of intact proteins (6 – 669 kDa) was achieved in less than 7 min with high-resolution and high efficiency. More importantly, we have shown that this UHP-SEC provides high-resolution separation of intact proteins using a MS-friendly volatile solvent system, allowing the direct top-down MS analysis of SEC eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP-SEC is an attractive LC strategy for the size-separation of proteins with great potential for top-down proteomics.
Protein separation; liquid chromatography; mass spectrometry; UPLC; post-translational modifications
Tropomyosins (Tms) are a family of highly conserved actin-binding proteins that play critical roles in a variety of processes, most notably, in the regulation of muscle contraction and relaxation. It is well known that different Tm isoforms have distinct functions and that altered expression of Tm isoforms could lead to changes in cardiac structure and function. To precisely define Tm isoform expression in the human heart, towards a better understanding of their functional roles, we have employed top-down mass spectrometry for in-depth proteomic characterization of Tm isoforms. Using a minimal amount of human heart tissue from rejected donor organs, we confirmed the presence of multiple Tm isoforms including α-Tm, β-Tm and κ-Tm in the human heart, with α-Tm being the predominant isoform, followed by minor isoforms of β-Tm and κ-Tm. Interestingly, our data revealed regional variations of Tm isoforms and posttranslational modifications in the human heart. Specifically, the expression level of κ-Tm was highest in the left atrium but nearly undetectable in the left ventricle. The phosphorylation level of α-Tm (pα-Tm) was significantly higher in the atria than it was in the ventricles. The sequences of all Tm isoforms were characterized and the sites of post-translational modifications were localized. Clearly, top-down mass spectrometry is an attractive method for comprehensive characterization of Tm isoforms and post-translational modifications since it can universally detect and quantify all types of protein modifications without a priori knowledge and without the need for specific antibodies.
Tropomyosin; Muscle Contraction; Isoforms; Mass Spectrometry; Post-translational modification
Lysine acetylation has emerged as a major posttranslational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion, and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely-related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.
We report a method based on thiol-ene chemistry that enables the synthesis and purification of ubiquitin oligomers with ≥ 4 units. This is the first time in which a free-radical polymerization is used to construct oligomers that functionally mimic natural biopolymers. This approach can be applied towards the synthesis of 6-linked ubiquitin oligomers currently inaccessible by enzymatic methods. Using these chains, one can study their roles in the ubiquitin proteasome system and the DNA damage response pathway.
ubiquitin; site-specific modification; thiol-ene polymerization
Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography–mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF.
Cardiac troponin I (cTnI) is the current standard biomarker for diagnosing acute myocardial infarction and for risk-stratification of acute coronary syndromes in patients. However it remains unclear how the epitope specificity of antibodies in immunoassays influences the detection of various modified forms of cTnI.
Four mouse anti-human cTnI monoclonal antibodies targeting different regions of human cTnI were chosen for immunoaffinity purification of cTnI from human and swine cardiac tissue. High-resolution intact protein mass spectrometry was employed to assess the comparative performance of these four antibodies in detecting modified forms of cTnI.
Our data revealed that antibody selection significantly impacts the relative protein yield of cTn from immunoaffinity purification. Remarkably, a single amino acid variation in cTnI (G->S) in the epitope region completely abolished the binding between monoclonal antibody 560 and swine cTnI in solution. Moreover, proteolytic degradation around the epitope region severely compromised the detection of proteolytic fragment forms of cTnI by monoclonal antibodies. In contrast, the phosphorylation status near the epitope region did not significantly affect the antibody recognition of cTnI.
Caution needs to be taken in the interpretation of the data produced by immuno-assays with monoclonal antibodies against various epitopes of cTnI.
cardiac troponin; mass spectrometry; epitope; immunoassays; biomarker; acute myocardial infarction
Accumulating evidence indicates that the long non-coding RNA HOTAIR plays a critical role in cancer progression and metastasis. However, the overall biological role and clinical significance of HOTAIR in gastric carcinogenesis remains largely unknown.
HOTAIR expression was measured in 78 paired cancerous and noncancerous tissue samples by real-time PCR. The effects of HOTAIR on gastric cancer cells were studied by overexpression and RNA interference approaches in vitro and in vivo. Insights of the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatic analysis, luciferase assays and RNA binding protein immunoprecipitation (RIP). The positive HOTAIR/HER2 interaction was identified and verified by immunohistochemistry assay and bivariate correlation analysis.
HOTAIR upregulation was associated with larger tumor size, advanced pathological stage and extensive metastasis, and also correlated with shorter overall survival of gastric cancer patients. Furthermore, HOTAIR overexpression promoted the proliferation, migration and invasion of gastric carcinoma cells, while HOTAIR depletion inhibited both cell invasion and cell viability, and induced growth arrest in vitro and in vivo. In particular, HOTAIR may act as a ceRNA, effectively becoming a sink for miR-331-3p, thereby modulating the derepression of HER2 and imposing an additional level of post-transcriptional regulation. Finally, the positive HOTAIR/HER2 correlation was significantly associated with advanced gastric cancers.
HOTAIR overexpression represents a biomarker of poor prognosis in gastric cancer, and may confer malignant phenotype to tumor cells. The ceRNA regulatory network involving HOTAIR and the positive interaction between HOTAIR and HER2 may contribute to a better understanding of gastric cancer pathogenesis and facilitate the development of lncRNA-directed diagnostics and therapeutics against this disease.
Competing endogenous RNA; HER2; HOTAIR; Gastric cancer; Proliferation and invasion