Adam D. Catherman

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.