13 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
Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry
Top-down
electron capture dissociation (ECD) Fourier transform
ion cyclotron resonance (FTICR) mass spectrometry was performed for
structural analysis of an intact monoclonal antibody (IgG1kappa (κ)
isotype, ∼148 kDa). Simultaneous ECD for all charge states
(42+ to 58+) generates more extensive cleavages than ECD for an isolated
single charge state. The cleavages are mainly localized in the variable
domains of both heavy and light chains, the respective regions between
the variable and constant domains in both chains, the region between
heavy-chain constant domains C<sub>H</sub>2 and C<sub>H</sub>3, and
the disulfide bond (S–S)-linked heavy-chain constant domain
C<sub>H</sub>3. The light chain yields mainly N-terminal fragment
ions due to the protection of the interchain disulfide bond between
light and heavy chain, and limited cleavage sites are observed in
the variable domains for each chain, where the S–S spans the
polypeptide backbone. Only a few cleavages in the S–S-linked
light-chain constant domain, hinge region, and heavy-chain constant
domains C<sub>H</sub>1 and C<sub>H</sub>2 are observed, leaving glycosylation
uncharacterized. Top-down ECD with a custom-built 9.4 T FTICR mass
spectrometer provides more extensive sequence coverage for structural
characterization of IgG1κ than does top-down collision-induced
dissociation (CID) and electron transfer dissociation (ETD) with hybrid
quadrupole time-of-flight instruments and comparable sequence coverage
for top-down ETD with orbitrap mass analyzers
Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry
Top-down
electron capture dissociation (ECD) Fourier transform
ion cyclotron resonance (FTICR) mass spectrometry was performed for
structural analysis of an intact monoclonal antibody (IgG1kappa (κ)
isotype, ∼148 kDa). Simultaneous ECD for all charge states
(42+ to 58+) generates more extensive cleavages than ECD for an isolated
single charge state. The cleavages are mainly localized in the variable
domains of both heavy and light chains, the respective regions between
the variable and constant domains in both chains, the region between
heavy-chain constant domains C<sub>H</sub>2 and C<sub>H</sub>3, and
the disulfide bond (S–S)-linked heavy-chain constant domain
C<sub>H</sub>3. The light chain yields mainly N-terminal fragment
ions due to the protection of the interchain disulfide bond between
light and heavy chain, and limited cleavage sites are observed in
the variable domains for each chain, where the S–S spans the
polypeptide backbone. Only a few cleavages in the S–S-linked
light-chain constant domain, hinge region, and heavy-chain constant
domains C<sub>H</sub>1 and C<sub>H</sub>2 are observed, leaving glycosylation
uncharacterized. Top-down ECD with a custom-built 9.4 T FTICR mass
spectrometer provides more extensive sequence coverage for structural
characterization of IgG1κ than does top-down collision-induced
dissociation (CID) and electron transfer dissociation (ETD) with hybrid
quadrupole time-of-flight instruments and comparable sequence coverage
for top-down ETD with orbitrap mass analyzers
Effective Protein Separation by Coupling Hydrophobic Interaction and Reverse Phase Chromatography for Top-down Proteomics
One
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
proteomics
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
Online Hydrophobic Interaction Chromatography–Mass Spectrometry for Top-Down Proteomics
Recent progress in top-down proteomics
has led to a demand for
mass spectrometry (MS)-compatible chromatography techniques to separate
intact proteins using volatile mobile phases. Conventional hydrophobic
interaction chromatography (HIC) provides high-resolution separation
of proteins under nondenaturing conditions but requires high concentrations
of nonvolatile salts. Herein, we introduce a series of more-hydrophobic
HIC materials that can retain proteins using MS-compatible concentrations
of ammonium acetate. The new HIC materials appear to function as a
hybrid form of conventional HIC and reverse phase chromatography.
The function of the salt seems to be preserving protein structure
rather than promoting retention. Online HIC-MS is feasible for both
qualitative and quantitative analysis. This is demonstrated with standard
proteins and a complex cell lysate. The mass spectra of proteins from
the online HIC-MS exhibit low charge-state distributions, consistent
with those commonly observed in native MS. Furthermore, HIC-MS can
chromatographically separate proteoforms differing by minor modifications.
Hence, this new HIC-MS combination is promising for top-down proteomics
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
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