Top-down Protein Characterization Research Articles

Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry.

Tandem mass spectrometry of denatured, multiply charged high mass protein precursor ions yield extremely dense spectra with hundreds of broad and overlapping product ion isotopic distributions of differing charge states that yield an elevated baseline of unresolved “noise” centered about the precursor ion. Development of mass analyzers and signal processing methods to increase mass resolving power and manipulation of precursor and product ion charge through solution additives or ion–ion reactions have been thoroughly explored as solutions to spectral congestion. Here, we demonstrate the utility of electron capture dissociation (ECD) coupled with high-resolution cyclic ion mobility spectrometry (cIMS) to greatly increase top-down protein characterization capabilities. Congestion of protein ECD spectra was reduced using cIMS of the ECD product ions and “mobility fractions”, that is, extracted mass spectra for segments of the 2D mobiligram (m/z versus drift time). For small proteins, such as ubiquitin (8.6 kDa), where mass resolving power was not the limiting factor for characterization, pre-IMS ECD and mobility fractions did not significantly increase protein sequence coverage, but an increase in the number of identified product ions was observed. However, a dramatic increase in performance, measured by protein sequence coverage, was observed for larger and more highly charged species, such as the +35 charge state of carbonic anhydrase (29 kDa). Pre-IMS ECD combined with mobility fractions yielded a 135% increase in the number of annotated isotope clusters and a 75% increase in unique product ions compared to processing without using the IMS dimension. These results yielded 89% sequence coverage for carbonic anhydrase.

Top-down analysis of disulfide-linked proteins using photoinduced radical reactions and ET-DDC

Top-down characterization of proteins via tandem mass spectrometry (MS/MS) can be challenging due to the presence of multiple disulfide bond linkages; which significantly inhibit the backbone cleavage efficiency for the formation of structurally informative fragment ions. In this study, we present a strategy of pairing a solution-phase photoinitiating system with dipolar direct current induced collisional activation of electron transfer products (ET-DDC) of proteins for a top-down MS/MS approach. The photoinitiating system allows for a rapid scission of all the disulfide linkages in the protein (on the time scale of seconds) with high efficiency (near to complete reduction); while ET-DDC collisional activation improves the fragmentation efficiency for the protein via broadband activation of all the first-generation charge reduced precursor ions (e.g., electron transfer no-dissociation or ETnoD products) from electron transfer reactions over a wide mass-to-charge range. As a result, this approach enabled the generation of extensive sequence informative fragment ion yields for a rapid and enhanced structural characterization of disulfide-linked proteins.

A Top-Down Proteomics Platform Coupling Serial Size Exclusion Chromatography and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Mass spectrometry (MS) based top-down proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S: N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30-50 kDa) simultaneously in a single mass spectrum within a 100 m/ z window. Notably, within a 5 m/ z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30-50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures.

Top-Down Characterization of Proteins with Intact Disulfide Bonds Using Activated-Ion Electron Transfer Dissociation.

Here we report the fragmentation of disulfide linked intact proteins using activated-ion electron transfer dissociation (AI-ETD) for top-down protein characterization. This fragmentation method is then compared to the alternative methods of beam-type collisional activation (HCD), electron transfer dissociation (ETD), and electron transfer and higher-energy collision dissociation (EThcD). We analyzed multiple precursor charge states of the protein standards bovine insulin, α-lactalbumin, lysozyme, β-lactoglobulin, and trypsin inhibitor. In all cases, we found that AI-ETD provides a boost in protein sequence coverage information and the generation of fragment ions from within regions enclosed by disulfide bonds. AI-ETD shows the largest improvement over the other techniques when analyzing highly disulfide linked and low charge density precursor ions. This substantial improvement is attributed to the concurrent irradiation of the gas phase ions while the electron-transfer reaction is taking place, mitigating nondissociative electron transfer, helping unfold the gas phase protein during the electron transfer event, and preventing disulfide bond reformation. We also show that AI-ETD is able to yield comparable sequence coverage information when disulfide bonds are left intact relative to proteins that have been reduced and alkylated. This work demonstrates that AI-ETD is an effective fragmentation method for the analysis of proteins with intact disulfide bonds, dramatically enhancing sequence ion generation and total sequence coverage compared to HCD and ETD.

Solid-phase extraction strategies to surmount body fluid sample complexity in high-throughput mass spectrometry-based proteomics.

For large-scale and standardized applications in mass spectrometry- (MS-) based proteomics automation of each step is essential. Here we present high-throughput sample preparation solutions for balancing the speed of current MS-acquisitions and the time needed for analytical workup of body fluids. The discussed workflows reduce body fluid sample complexity and apply for both bottom-up proteomics experiments and top-down protein characterization approaches. Various sample preparation methods that involve solid-phase extraction (SPE) including affinity enrichment strategies have been automated. Obtained peptide and protein fractions can be mass analyzed by direct infusion into an electrospray ionization (ESI) source or by means of matrix-assisted laser desorption ionization (MALDI) without further need of time-consuming liquid chromatography (LC) separations.

Nano-LC FTICR Tandem Mass Spectrometry for Top-Down Proteomics: Routine Baseline Unit Mass Resolution of Whole Cell Lysate Proteins up to 72 kDa

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.

Top‐down protein characterization facilitated by ion/ion reactions on a quadrupole/time of flight platform

In comparison to bottom-up proteomics approaches, whereby peptides derived from proteolytic digestion are analyzed, top-down approaches, involving direct analysis of intact proteins, provide higher specificity for protein identification and are better-suited for the characterization of sequence variants. However, top-down protein characterization usually requires more sophisticated instrumentation and methodologies to deal with the more complex tandem mass spectra derived from dissociation of high mass multiply charged intact proteins. Gas-phase ion/ion reactions are universally applicable and have proved to be useful in mixture analysis and top-down biomolecule characterization. The coupling of the ion/ion proton transfer reaction in the context of MS/MS has been demonstrated to expand informing power in top-down protein characterization, particularly with platforms that employ electrodynamic ion trap and TOF mass analysis. In addition, probing protein primary structure using ion/ion electron transfer dissociation usually provides extensive structurally informative fragmentation and also allows for the localization of labile PTMs. Here, the performance of the widely used quadrupole/TOF platform, equipped with ion/ion reaction functionality, for top-down protein characterization is summarized, and various methodologies employing ion/ion reactions are reviewed.

Simultaneous transmission mode collision-induced dissociation and ion/ion reactions for top-down protein identification/characterization using a quadrupole/time-of-flight tandem mass spectrometer.

Simultaneous transmission mode collision-induced dissociation (CID) and ion/ion proton transfer reactions have been implemented on a quadrupole/time-of-flight (TOF) tandem mass spectrometer. Reagent anions were trapped in a pressurized quadrupole collision cell by applying appropriate dc voltages while multiply protonated protein precursor ions were injected into the collision cell at energies sufficient to give rise to CID. Intact precursor ions as well as fragment ions underwent ion/ion proton transfer reactions during their passage through the collision cell and on to an orthogonal acceleration TOF mass analyzer. The resulting product ion spectrum was then submitted to deconvolution to yield a "zero-charge" spectrum, which was then matched against in silico produced spectra derived from a protein database. Dramatic improvements in the scores associated with correct matches were obtained relative to CID data without the benefit of ion/ion reactions for proteins as large as carbonic anhydrase (29 kDa). The parameters that most affect the extent of ion/ion proton transfer during transmission through the instrument include the number of anions stored in the collision cell, the amplitude of the radio frequency trapping voltage, the voltage of the LINAC potential associated with the collision cell, and the collision gas pressure. This work demonstrates that it is possible to effect whole protein tandem mass spectrometry with simultaneous CID, ion/ion reactions, and mass analysis for high duty cycle top-down protein characterization.

Top-Down Identification and Quantification of Stable Isotope Labeled Proteins from Aspergillus flavus Using Online Nano-Flow Reversed-Phase Liquid Chromatography Coupled to a LTQ-FTICR Mass Spectrometer

Online liquid chromatography-mass spectrometric (LC-MS) analysis of intact proteins (i.e., top-down proteomics) is a growing area of research in the mass spectrometry community. A major advantage of top-down MS characterization of proteins is that the information of the intact protein is retained over the vastly more common bottom-up approach that uses protease-generated peptides to search genomic databases for protein identification. Concurrent to the emergence of top-down MS characterization of proteins has been the development and implementation of the stable isotope labeling of amino acids in cell culture (SILAC) method for relative quantification of proteins by LC-MS. Herein we describe the qualitative and quantitative top-down characterization of proteins derived from SILAC-labeled Aspergillus flavus using nanoflow reversed-phase liquid chromatography directly coupled to a linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FTICR-MS). A. flavus is a toxic filamentous fungus that significantly impacts the agricultural economy and human health. SILAC labeling improved the confidence of protein identification, and we observed 1318 unique protein masses corresponding to 659 SILAC pairs, of which 22 were confidently identified. However, we have observed some limiting issues with regard to protein quantification using top-down MS/MS analyses of SILAC-labeled proteins. The role of SILAC labeling in the presence of competing endogenously produced amino acid residues and its impact on quantification of intact species are discussed in detail.

Emerging methods in proteomics: top-down protein characterization by multistage tandem mass spectrometry

"Top-down" mass spectrometry methods have emerged as an attractive alternative to conventional "bottom-up" approaches for the comprehensive characterization of co- and post-translational protein modifications. Here we present a brief overview of current strategies employed for top-down protein characterization and discuss the key technical challenges and solutions associated with their implementation on a range of mass spectrometry instrument platforms. For more specific details regarding the individual strategies described herein, interested readers are referred to the references cited at the end of this article.

Infrared Multiphoton Dissociation in Quadrupole Time-of-Flight Mass Spectrometry: Top-Down Characterization of Proteins

The first implementation of infrared multiphoton dissociation (IRMPD) for a hybrid quadrupole time-of-flight (QqTOF) mass spectrometer is reported. Ions were trapped in the radio frequency-only quadrupole (q2), which normally serves as a collision cell, and irradiated by a continuous CO2 IR laser. The laser beam was introduced coaxially with the quadrupoles in order to maximize overlap with the ion path. The resolution of the TOF mass analyzer allowed direct charge state determination for fragments smaller than 7 kDa. For larger fragments, the charge state could be assigned using the multiple losses of water, characteristic for IRMPD of proteins. The analytical performance is demonstrated by top-down sequencing of several representative proteins (equine myoglobin, bovine casein, and human insulin and chaperonin 10). Various post-translational modifications such as phosphorylation, acetylation, formation of disulfide bridges, and removal of N-terminal methionine followed by acetylation are detected and characterized. The utility of IRMPD for the analysis of biological samples is demonstrated in a study of a recently identified potential marker for endometrial cancer, chaperonin 10.