18 research outputs found
MASH Suite Pro: A Comprehensive Software Tool for Top-Down Proteomics
Top-down mass spectrometry (MS)-based proteomics is arguably a disruptive technology for the comprehensive analysis of all proteoforms arising from genetic variation, alternative splicing, and posttranslational modifications (PTMs). However, the complexity of top-down high-resolution mass spectra presents a significant challenge for data analysis. In contrast to the well-developed software packages available for data analysis in bottom-up proteomics, the data analysis tools in top-down proteomics remain underdeveloped. Moreover, despite recent efforts to develop algorithms and tools for the deconvolution of top-down high-resolution mass spectra and the identification of proteins from complex mixtures, a multifunctional software platform, which allows for the identification, quantitation, and characterization of proteoforms with visual validation, is still lacking. Herein, we have developed MASH Suite Pro, a comprehensive software tool for top-down proteomics with multifaceted functionality. MASH Suite Pro is capable of processing high-resolution MS and tandem MS (MS/MS) data using two deconvolution algorithms to optimize protein identification results. In addition, MASH Suite Pro allows for the characterization of PTMs and sequence variations, as well as the relative quantitation of multiple proteoforms in different experimental conditions. The program also provides visualization components for validation and correction of the computational outputs. Furthermore, MASH Suite Pro facilitates data reporting and presentation via direct output of the graphics. Thus, MASH Suite Pro significantly simplifies and speeds up the interpretation of high-resolution top-down proteomics data by integrating tools for protein identification, quantitation, characterization, and visual validation into a customizable and user-friendly interface. We envision that MASH Suite Pro will play an integral role in advancing the burgeoning field of top-down proteomics
SR Protein Kinases Regulate the Splicing of Cardiomyopathy-Relevant Genes via Phosphorylation of the RSRSP Stretch in RBM20
(1) Background: RNA binding motif 20 (RBM20) regulates mRNA splicing specifically in muscle tissues. Missense mutations in the arginine/serine (RS) domain of RBM20 lead to abnormal gene splicing and have been linked to severe dilated cardiomyopathy (DCM) in human patients and animal models. Interestingly, many of the reported DCM-linked missense mutations in RBM20 are in a highly conserved RSRSP stretch within the RS domain. Recently, it was found that the two Ser residues within this stretch are constitutively phosphorylated, yet the identity of the kinase(s) responsible for phosphorylating these residues, as well as the function of RSRSP phosphorylation, remains unknown. (2) Methods: The ability of three known SR protein kinases (SRPK1, CLK1, and AKT2) to phosphorylate the RBM20 RSRSP stretch and regulate target gene splicing was evaluated by using both in vitro and in vivo approaches. (3) Results: We found that all three kinases phosphorylated S638 and S640 in the RSRSP stretch and regulated RBM20 target gene splicing. While SRPK1 and CLK1 were both capable of directly phosphorylating the RS domain in RBM20, whether AKT2-mediated control of the RS domain phosphorylation is direct or indirect could not be determined. (4) Conclusions: Our results indicate that SR protein kinases regulate the splicing of a cardiomyopathy-relevant gene by modulating phosphorylation of the RSRSP stretch in RBM20. These findings suggest that SR protein kinases may be potential targets for the treatment of RBM20 cardiomyopathy
Three Dimensional Liquid Chromatography Coupling Ion Exchange Chromatography/Hydrophobic Interaction Chromatography/Reverse Phase Chromatography for Effective Protein Separation in Top-Down Proteomics
To address the complexity of the
proteome in mass spectrometry
(MS)-based top-down proteomics, multidimensional liquid chromatography
(MDLC) strategies that can effectively separate proteins with high
resolution and automation are highly desirable. Although various MDLC
methods that can effectively separate peptides from protein digests
exist, very few MDLC strategies, primarily consisting of 2DLC, are
available for intact protein separation, which is insufficient to
address the complexity of the proteome. We recently demonstrated that
hydrophobic interaction chromatography (HIC) utilizing a MS-compatible
salt can provide high resolution separation of intact proteins for
top-down proteomics. Herein, we have developed a novel 3DLC strategy
by coupling HIC with ion exchange chromatography (IEC) and reverse
phase chromatography (RPC) for intact protein separation. We demonstrated
that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional
2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions)
from a crude HEK 293 cell lysate, a total of 640 proteins were identified
in the 3D approach (corresponding to 201 nonredundant proteins) as
compared to 47 in the 2D approach, whereas simply prolonging the gradients
in RPC in the 2D approach only led to minimal improvement in protein
separation and identifications. Therefore, this novel 3DLC method
has great potential for effective separation of intact proteins to
achieve deep proteome coverage in top-down proteomics
Impact of Phosphorylation on the Mass Spectrometry Quantification of Intact Phosphoproteins
Protein phosphorylation
is a ubiquitous and critical post-translational
modification (PTM) involved in numerous cellular processes. Mass spectrometry
(MS)-based proteomics has emerged as the preferred technology for
protein identification, characterization, and quantification. Whereas
ionization/detection efficiency of peptides in electrospray ionization
(ESI)-MS are markedly influenced by the presence of phosphorylation,
the physicochemical properties of intact proteins are assumed not
to vary significantly due to the relatively smaller modification on
large intact proteins. Thus, the ionization/detection efficiency of
intact phosphoprotein is hypothesized not to alter appreciably for
subsequent MS quantification. However, this hypothesis has never been
rigorously tested. Herein, we systematically investigated the impact
of phosphorylation on ESI-MS quantification of mono- and multiply
phosphorylated proteins. We verified that a single phosphorylation
did not appreciably affect the ESI-MS quantification of phosphoproteins
as demonstrated in the enigma homolog isoform 2 (28 kDa) with monophosphorylation.
Moreover, different ionization and desolvation parameters did not
impact phosphoprotein quantification. In contrast to monophosphorylation,
multiphosphorylation noticeably affected ESI-MS quantification of
phosphoproteins likely due to differential ionization/detection efficiency
between unphosphorylated and phosphorylated proteoforms as shown in
the pentakis-phosphorylated β-casein (24 kDa)
Specific Enrichment of Phosphoproteins Using Functionalized Multivalent Nanoparticles
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 toward a comprehensive analysis of the phosphoproteome
Quantitative Proteomics and Immunohistochemistry Reveal Insights into Cellular and Molecular Processes in the Infarct Border Zone One Month after Myocardial Infarction
Postinfarction
remodeling and expansion of the peri-infarct border
zone (BZ) directly correlate with mortality following myocardial infarction
(MI); however, the cellular and molecular mechanisms underlying remodeling
processes in the BZ remain unclear. Herein, we utilized a label-free
quantitative proteomics approach in combination with immunohistochemical
analyses to gain a better understanding of processes contributing
to postinfarction remodeling of the peri-infarct BZ in a swine model
of MI with reperfusion. Our analysis uncovered a significant down-regulation
of proteins involved in energy metabolism, indicating impaired myocardial
energetics and possibly mitochondrial dysfunction, in the peri-scar
BZ. An increase in endothelial and vascular smooth muscles cells,
as well as up-regulation of proteins implicated in vascular endothelial
growth factor (VEGF) signaling and marked changes in the expression
of extracellular matrix and subendothelial basement membrane proteins,
is indicative of active angiogenesis in the infarct BZ. A pronounced
increase in macrophages in the peri-infarct BZ was also observed,
and proteomic analysis uncovered evidence of persistent inflammation
in this tissue. Additional evidence suggested an increase in cellular
proliferation that, concomitant with increased nestin expression,
indicates potential turnover of endogenous stem cells in the BZ. A
marked up-regulation of pro-apoptotic proteins, as well as the down-regulation
of proteins important for adaptation to mechanical, metabolic, and
oxidative stress, likely contributes to increased apoptosis in the
peri-infarct BZ. The cellular processes and molecular pathways identified
herein may have clinical utility for therapeutic intervention aimed
at limiting remodeling and expansion of the BZ myocardium and preventing
the development of heart failure post-MI
Top-down Proteomics Reveals Concerted Reductions in Myofilament and Z-disc Protein Phosphorylation after Acute Myocardial Infarction
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
New Mass-Spectrometry-Compatible Degradable Surfactant for Tissue Proteomics
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
MS-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