Mapping intact protein isoforms in discovery mode using top-down proteomics.

A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry. This 'bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research.

Comparative top down proteomics of peripheral blood mononuclear cells from kidney transplant recipients with normal kidney biopsies or acute rejection.

Recent studies utilizing transcriptomics, metabolomics, and bottom up proteomics have identified molecular signatures of kidney allograft pathology. Although these results make significant progress toward non-invasive differential diagnostics of dysfunction of a transplanted kidney, they provide little information on the intact, often modified, protein molecules present during progression of this pathology. Because intact proteins underpin diverse biological processes, measuring the relative abundance of their modified forms promises to advance mechanistic understanding, and might provide a new class of biomarker candidates. Here, we used top down proteomics to inventory the modified forms of whole proteins in peripheral blood mononuclear cells (PBMCs) taken at the time of kidney biopsy for 40 kidney allograft recipients either with healthy transplants or those suffering acute rejection. Supported by gas-phase fragmentation of whole protein ions during tandem mass spectrometry, we identified 344 proteins mapping to 2905 distinct molecular forms (proteoforms). Using an initial implementation of a label-free approach to quantitative top down proteomics, we obtained evidence suggesting relative abundance changes in 111 proteoforms between the two patient groups. Collectively, our work is the first to catalog intact protein molecules in PBMCs and suggests differentially abundant proteoforms for further analysis.

Defining the Neuropeptidome of the Spiny Lobster Panulirus interruptus Brain Using a Multidimensional Mass Spectrometry-Based Platform.

Decapod crustaceans are important animal models for neurobiologists due to their relatively simple nervous systems with well-defined neural circuits and extensive neuromodulation by a diverse set of signaling peptides. However, biochemical characterization of these endogenous neuropeptides is often challenging due to limited sequence information about these neuropeptide genes and the encoded preprohormones. By taking advantage of sequence homology in neuropeptides observed in related species using a home-built crustacean neuropeptide database, we developed a semi-automated sequencing strategy to characterize the neuropeptidome of Panulirus interruptus, an important aquaculture species, with few known neuropeptide preprohormone sequences. Our streamlined process searched the high mass accuracy and high-resolution data acquired on a LTQ-Orbitrap with a flexible algorithm in ProSight that allows for sequence discrepancy from reported sequences in our database, resulting in the detection of 32 neuropeptides, including 19 novel ones. We further improved the overall coverage to 51 neuropeptides with our multidimensional platform that employed multiple analytical techniques including dimethylation-assisted fragmentation, de novo sequencing using nanoliquid chromatography-electrospray ionization-quadrupole-time-of-flight (nanoLC-ESI-Q-TOF), direct tissue analysis, and mass spectrometry imaging on matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF. The high discovery rate from this unsequenced model organism demonstrated the utility of our neuropeptide discovery pipeline and highlighted the advantage of utilizing multiple sequencing strategies. Collectively, our study expands the catalog of crustacean neuropeptides and more importantly presents an approach that can be adapted to exploring neuropeptidome from species that possess limited sequence information.

Native GELFrEE: a new separation technique for biomolecular assemblies.

The cadre of protein complexes in cells performs an array of functions necessary for life. Their varied structures are foundational to their ability to perform biological functions, lending great import to the elucidation of complex composition and dynamics. Native separation techniques that are operative on low sample amounts and provide high resolution are necessary to gain valuable data on endogenous complexes. Here, we detail and optimize the use of tube gel separations to produce samples proven compatible with native, multistage mass spectrometry (nMS/MS). We find that a continuous system (i.e., no stacking gel) with a gradient in its extent of cross-linking and use of the clear native buffer system performs well for both fractionation and native mass spectrometry of heart extracts and a fungal secretome. This integrated advance in separations and nMS/MS offers the prospect of untargeted proteomics at the next hierarchical level of protein organization in biology.

Large-scale top-down proteomics of the human proteome: membrane proteins, mitochondria, and senescence.

Top-down proteomics is emerging as a viable method for the routine identification of hundreds to thousands of proteins. In this work we report the largest top-down study to date, with the identification of 1,220 proteins from the transformed human cell line H1299 at a false discovery rate of 1%. Multiple separation strategies were utilized, including the focused isolation of mitochondria, resulting in significantly improved proteome coverage relative to previous work. In all, 347 mitochondrial proteins were identified, including ~50% of the mitochondrial proteome below 30 kDa and over 75% of the subunits constituting the large complexes of oxidative phosphorylation. Three hundred of the identified proteins were found to be integral membrane proteins containing between 1 and 12 transmembrane helices, requiring no specific enrichment or modified LC-MS parameters. Over 5,000 proteoforms were observed, many harboring post-translational modifications, including over a dozen proteins containing lipid anchors (some previously unknown) and many others with phosphorylation and methylation modifications. Comparison between untreated and senescent H1299 cells revealed several changes to the proteome, including the hyperphosphorylation of HMGA2. This work illustrates the burgeoning ability of top-down proteomics to characterize large numbers of intact proteoforms in a high-throughput fashion.

Fragmentation of integral membrane proteins in the gas phase.

Integral membrane proteins (IMPs) are of great biophysical and clinical interest because of the key role they play in many cellular processes. Here, a comprehensive top down study of 152 IMPs and 277 soluble proteins from human H1299 cells including 11 087 fragments obtained from collisionally activated dissociation (CAD), 6452 from higher-energy collisional dissociation (HCD), and 2981 from electron transfer dissociation (ETD) shows their great utility and complementarity for the identification and characterization of IMPs. A central finding is that ETD is ∼2-fold more likely to cleave in soluble regions than threshold fragmentation methods, whereas the reverse is observed in transmembrane domains with an observed ∼4-fold bias toward CAD and HCD. The location of charges just prior to dissociation is consistent with this directed fragmentation: protons remain localized on basic residues during ETD but easily mobilize along the backbone during collisional activation. The fragmentation driven by these protons, which is most often observed in transmembrane domains, both is of higher yield and occurs over a greater number of backbone cleavage sites. Further, while threshold dissociation events in transmembrane domains are on average 10.1 (CAD) and 9.2 (HCD) residues distant from the nearest charge site (R, K, H, N-terminus), fragmentation is strongly influenced by the N- or C-terminal position relative to that site: the ratio of observed b- to y-fragments is ∼1:3 if the cleavage occurs >7 residues N-terminal and ∼3:1 if it occurs >7 residues C-terminal to the nearest basic site. Threshold dissociation products driven by a mobilized proton appear to be strongly dependent on not only relative position of a charge site but also N- or C-terminal directionality of proton movement.

Top Down proteomics: facts and perspectives.

The rise of the "Top Down" method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious "inference" problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years.

High-definition de novo sequencing of crustacean hyperglycemic hormone (CHH)-family neuropeptides.

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8-9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.

Top down proteomics of human membrane proteins from enriched mitochondrial fractions.

The interrogation of intact integral membrane proteins has long been a challenge for biological mass spectrometry. Here, we demonstrate the application of top down mass spectrometry to whole membrane proteins below 60 kDa with up to 8 transmembrane helices. Analysis of enriched mitochondrial membrane preparations from human cells yielded identification of 83 integral membrane proteins, along with 163 membrane-associated or soluble proteins, with a median q value of 3 × 10(-10). An analysis of matching fragment ions demonstrated that significantly more fragment ions were found within transmembrane domains than would be expected based upon the observed protein sequence. In total, 46 proteins from the complexes of oxidative phosphorylation were identified which exemplifies the increasing ability of top down proteomics to provide extensive coverage in a biological network.

Analysis of intact protein isoforms by mass spectrometry.

The diverse proteome of an organism arises from such events as single nucleotide substitutions at the DNA level, different RNA processing, and dynamic enzymatic post-translational modifications. This minireview focuses on the measurement of intact proteins to describe the diversity found in proteomes. The field of biological mass spectrometry has steadily advanced, enabling improvements in the characterization of single proteins to proteins derived from cells or tissues. In this minireview, we discuss the basic technology for "top-down" intact protein analysis. Furthermore, examples of studies involved with the qualitative and quantitative analysis of full-length polypeptides are provided.

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.

Robust analysis of the yeast proteome under 50 kDa by molecular-mass-based fractionation and top-down mass spectrometry.

As the process of top-down mass spectrometry continues to mature, we benchmark the next installment of an improving methodology that incorporates a tube-gel electrophoresis (TGE) device to separate intact proteins by molecular mass. Top-down proteomics is accomplished in a robust fashion to yield the identification of hundreds of unique proteins, many of which correspond to multiple protein forms. The TGE platform separates 0-50 kDa proteins extracted from the yeast proteome into 12 fractions prior to automated nanocapillary LC-MS/MS in technical triplicate. The process may be completed in less than 72 h. From this study, 530 unique proteins and 1103 distinct protein species were identified and characterized, thus representing the highest coverage to date of the Saccharomyces cerevisiae proteome using top-down proteomics. The work signifies a significant step in the maturation of proteomics based on direct measurement and fragmentation of intact proteins.

Investigating the effects of conductivity on zone overlap with EMMA: computer simulation and experiment.

In this paper, we demonstrate, using both experiment and simulation, how sample zone conductivity can affect plug-plug mixing in small molecule applications of electrophoretically mediated microanalysis (EMMA). The effectiveness of in-line mixing, which is driven by potential, can vary widely with experimental conditions. Using two small molecule systems, the effects of local conductivity differences between analyte plugs, reagent plugs and the BGE on EMMA analyses are examined. Simul 5.0, a dynamic simulation program for CE systems, is used to understand the ionic boundaries and profiles that give rise to the experimentally obtained data for EMMA analyses for (i) creatinine determination via the Jaffe reaction, a reaction involving a neutral and an anion, and (ii) the redox reaction between gallate and 2,6-dichloroindophenol, two anions. Low sample conductivity, which is widely used in CE analyses, can be detrimental for in-line reactions involving a neutral reactant, as rapid migration of the ionic component across a low conductivity neutral zone results in poor reagent plug overlap and low reaction efficiency. Conversely, with two similarly charged reagents, a low conductivity sample plug is advantageous, as it allows field-amplified stacking of the reagents into a tight reaction zone. In addition, the complexity of simultaneously overlapping three reagent zones is considered, and experimental results validate the predictions made by the simulation. The simulations, however, do not appear to predict all of the observed experimental behavior. Overall, by combining experiment with simulation, an enhanced appreciation for the local field effects in EMMA is realized, and general guidelines for an advantageous sample matrix can be established for categories of EMMA analyses.

Intact mass detection, interpretation, and visualization to automate Top-Down proteomics on a large scale.

Applying high-throughput Top-Down MS to an entire proteome requires a yet-to-be-established model for data processing. Since Top-Down is becoming possible on a large scale, we report our latest software pipeline dedicated to capturing the full value of intact protein data in automated fashion. For intact mass detection, we combine algorithms for processing MS1 data from both isotopically resolved (FT) and charge-state resolved (ion trap) LC-MS data, which are then linked to their fragment ions for database searching using ProSight. Automated determination of human keratin and tubulin isoforms is one result. Optimized for the intricacies of whole proteins, new software modules visualize proteome-scale data based on the LC retention time and intensity of intact masses and enable selective detection of PTMs to automatically screen for acetylation, phosphorylation, and methylation. Software functionality was demonstrated using comparative LC-MS data from yeast strains in addition to human cells undergoing chemical stress. We further these advances as a key aspect of realizing Top-Down MS on a proteomic scale.

Size-sorting combined with improved nanocapillary liquid chromatography-mass spectrometry for identification of intact proteins up to 80 kDa.

Despite the availability of ultra-high-resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for online LC-MS to drive high-throughput top-down proteomics in a fashion similar to that of bottom-up proteomics. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary LC, with detection of proteins >50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier transform mass spectrometer. Protein identification was enabled by nozzle-skimmer dissociation and detection of fragment ions with <10 ppm mass accuracy for highly specific database searching using tailored software. This overall approach led to identification of proteins up to 80 kDa, with 10-60 proteins identified in single LC-MS runs of samples from yeast and human cell lines prefractionated by their molecular mass using a gel-based sieving system.

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis.

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

The emergence of top-down proteomics in clinical research.

Proteomic technology has advanced steadily since the development of 'soft-ionization' techniques for mass-spectrometry-based molecular identification more than two decades ago. Now, the large-scale analysis of proteins (proteomics) is a mainstay of biological research and clinical translation, with researchers seeking molecular diagnostics, as well as protein-based markers for personalized medicine. Proteomic strategies using the protease trypsin (known as bottom-up proteomics) were the first to be developed and optimized and form the dominant approach at present. However, researchers are now beginning to understand the limitations of bottom-up techniques, namely the inability to characterize and quantify intact protein molecules from a complex mixture of digested peptides. To overcome these limitations, several laboratories are taking a whole-protein-based approach, in which intact protein molecules are the analytical targets for characterization and quantification. We discuss these top-down techniques and how they have been applied to clinical research and are likely to be applied in the near future. Given the recent improvements in mass-spectrometry-based proteomics and stronger cooperation between researchers, clinicians and statisticians, both peptide-based (bottom-up) strategies and whole-protein-based (top-down) strategies are set to complement each other and help researchers and clinicians better understand and detect complex disease phenotypes.

The emerging process of Top Down mass spectrometry for protein analysis: biomarkers, protein-therapeutics, and achieving high throughput.

Top Down mass spectrometry (MS) has emerged as an alternative to common Bottom Up strategies for protein analysis. In the Top Down approach, intact proteins are fragmented directly in the mass spectrometer to achieve both protein identification and characterization, even capturing information on combinatorial post-translational modifications. Just in the past two years, Top Down MS has seen incremental advances in instrumentation and dedicated software, and has also experienced a major boost from refined separations of whole proteins in complex mixtures that have both high recovery and reproducibility. Combined with steadily advancing commercial MS instrumentation and data processing, a high-throughput workflow covering intact proteins and polypeptides up to 70 kDa is directly visible in the near future.

Determination of total antioxidant capacity of commercial beverage samples by capillary electrophoresis via inline reaction with 2,6-dichlorophenolindophenol.

This paper demonstrates proof-of-concept for the use of electrophoretically mediated microanalysis (EMMA) as a new approach to the determination of total antioxidant capacity (TAC). EMMA is a low-volume, high-efficiency capillary electrophoretic technique that has to date been underutilized for small molecule reactions. Here, nanoliter volumes of 2,6-dichlorophenolindophenol (DCIP) reagent solution are mixed with an antioxidant-containing sample within the confines of a narrow-bore capillary tube. The mixing is accomplished by exploiting differential migration rates of the reagents when a voltage field is applied across the length of the capillary tube. The ensuing electron transfer reaction between DCIP and the antioxidant(s) is then used as a quantitative measure of the TAC of the sample. Linear calibration using either redox form of DCIP is accomplished with standard solutions of ascorbic acid. Several commercial beverage samples are analyzed, and the TAC values obtained with the reported methodology are compared to results obtained with the widely used ferric reducing antioxidant power (FRAP) spectroscopic method. For the analysis of real samples of unknown ionic strength, the method of standard additions is shown to be superior to the use of external calibration. This easily automated EMMA method may represent a useful new approach to TAC determination.

A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome.

For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The "GELFrEE" (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5-25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.