Review
Top Down proteomics: Facts and perspectives

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Highlights

Abstract

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.

Section snippets

Proteomics in a post-genomics world

The rise in genome sequencing has greatly propelled the understanding of the living world, but alone is insufficient for full description of a biological system [1]. Focusing on the protein level, proteomics has emerged as another large-scale platform for improving the understanding of biology. Proteomic experiments can be used for the annotation and correction of genome sequences, quantitation of protein abundance, detection of post-translational modifications (PTMs), and identification of

Top Down proteomics

While a variety of methods, including cell imaging and protein arrays, are capable of large-scale protein study, mass spectrometry-based approaches are uniquely well suited in terms of throughput and sensitivity to handle proteome-wide investigations [2]. Mass spectrometry-based proteomics has traditionally been carried out in a Bottom Up approach [3], [4]. This entails the chemical or enzymatic digestion of proteins prior to their introduction to the mass spectrometer. The detection and

Intact protein separation methods

The great complexity within most proteomic samples requires that they be fractionated prior to introduction to the mass spectrometer [12]. Many separation strategies can be applied off-line, or independent of the mass spectrometer [13]. This entails collection of the eluted fractions followed by their infusion into the mass spectrometer. Using this approach, more instrument time can be spent collecting data on a single protein or simple mixture. Additionally, off-line separations are more

Liquid chromatography

One of the most common methods for the separation of intact proteins, peptides, and small molecules is liquid chromatography (LC). This general separation approach relies on differential partitioning of analytes between a liquid mobile phase and a stationary phase. In many cases, liquid chromatography can often be coupled to electrospray ionization (ESI), proving an effective method for on-line analysis [14]. While a variety of liquid chromatography methods have been developed, reversed-phase

Reversed-phase liquid chromatography

RPLC uses a non-polar stationary phase and a polar mobile phase, allowing the most hydrophilic analytes to elute first. Alkyl chains (C4, C5, C8, C18) linked to porous silica particles are common stationary phases, where shorter chains are generally preferred for intact proteins as these phases are less retentive and offer higher recovery [13]. Additionally, many reports have been published using derivatized nonporous silica (NPS) particles, which offer increased speed and protein recovery, but

Hydrophobic interaction liquid chromatography

In contrast with RPLC, HILIC utilizes a polar stationary phase and gradients of increasing water content, resulting in the elution of more hydrophobic species first [26], [27]. Analytes partition between the mobile phase and water-enriched region surrounding the stationary phase, differing from traditional normal phase chromatography where analytes are actually adsorbed to the hydrophilic stationary phase. Membrane proteins extracted from bovine heart mitochondria have been fractionated using

Ion exchange

While separation in RPLC and HILIC rely primarily on differences in hydrophobicity to achieve separation, ion-exchange chromatography (IEX) uses differences in the charge of the analyte. Increasing the ionic strength of the mobile phase is used to elute analytes from the charged stationary phase. Opiteck et al. reported the use of cation exchange coupled to on-line RPLC for the two dimensional separation of the Escherichia coli proteome [33]. Besides increasing the fractionation power of the

Electrophoresis

In addition to chromatography, electrophoresis, which relies on the differential migration of proteins in an applied electric field, is an extremely popular general approach for separating intact proteins [2], [13], [39]. The most common electrophoretic method is SDS–PAGE, in which SDS-coated protein molecules migrate through a polyacrylamide gel matrix in an electric field achieving separation based largely on molecular weight [40]. This is commonly utilized in Bottom Up proteomics by

Tube gel electrophoresis

While traditional gel-based approaches are generally not applicable to Top Down proteomics, similar separation strategies have been applied. Continuous-elution gel electrophoresis utilizes a tube gel column to separate proteins which are then collected as they elute from the end of the gel column [16]. This approach was applied to the fractionation of the Saccharomyces cerevisiae proteome using an acid-labile surfactant (ALS) rather than SDS, as it could be degraded upon acidification, limiting

Isoelectric focusing

Isoelectric focusing (IEF) for Top Down proteomics is generally considered more difficult as proteins tend to precipitate at their isoelectric point, significantly reducing their recovery from the gel media [18]. The Rotofor device uses an IEF separation but within an open channel, where the pH gradient is formed through the use of carrier ampholytes in solution between an acidic anode and a basic cathode [53]. While precipitation can still be problematic, especially for hydrophobic proteins,

Capillary electrophoresis

Another electrophoretic technique used for the separation of intact proteins is capillary electrophoresis (CE). The small capillaries (<100 μm inner diameter) used within CE allow for high separation voltages (10–30 kV) without Joule heating, thereby reducing separation time and increasing peak capacity by limiting longitudinal diffusion [56], [57], [58]. Capillary zone electrophoresis (CZE), the simplest separation mode in CE, utilizes differences in the electrophoretic mobility of the analytes

Mass spectrometry of intact proteins

The detection and identification of intact proteins, especially on a proteome-wide level, depends on high performance mass spectrometers [3]. High resolution and mass accuracy are critical to separate and accurately assign spectral peaks arising from complex precursor spectra containing multiple intact proteoforms or fragmentation spectra containing hundreds of fragment ions. Extremely high resolution may be required to distinguish disulfide bridges (Δm = 2 Da), deamidation (Δm = 1 Da),

Fourier transform ion cyclotron resonance mass spectrometry

Fourier transform ion cyclotron resonance mass spectrometry relies on the excitation of an ion at its cyclotron frequency within a strong magnetic field [77], [78], [79]. This excitation creates a spatially coherent packet of ions, which orbit at an increased radius, allowing for detection by monitoring the image current on a detection plate. The detected signal, also termed a transient, is converted from the time domain to the frequency domain through a Fourier transform, and then to m/z

Orbitrap mass spectrometry

A new type of Fourier transform mass spectrometer was described in 2000, the Orbitrap mass analyzer [101]. This trap features a pair of axially symmetric electrodes: a central “spindle-like” electrode and an outer “barrel-like electrode”. In this electric field, ions rotate around the central electrode while oscillating down the length of the electrode. The frequency of these oscillations is proportional to (m/z)1/2. Image current on the outer electrodes is monitored and the resulting time

Data processing for Top Down proteomics

While powerful separation devices and mass spectrometers can be used together to generate data impressive in both quality and quantity, it must be adequately processed in order to identify and characterize proteoforms. As Top Down proteomics continues to increase in throughput and complexity of the samples analyzed, it is clear that a software platform must allow for fast, automated processing of raw data. ProSight PTM was the first search engine and web application designed for the

Native mass spectrometry

Current high-throughput Top Down workflows have proven extremely successful at identifying a large number of the proteins present in human cells, yet the great majority of these studies have denatured the proteins prior to their introduction into the mass spectrometer [118]. While these conditions are gentle enough to preserve many covalent PTMs, the potentially biologically relevant non-covalent protein–protein and protein–ligand interactions are mostly destroyed. Native size-exclusion

Conclusion

Top Down proteomics offers an alternative to digestion-based approaches, with the promise of full protein characterization on a proteome-wide scale. While the measurement of intact proteins presents many technical challenges, the field has seen tremendous advances in separations tools, mass spectrometry instrumentation, and data processing. There has been a clear trend towards miniaturization of separations and increased use on-line and multidimensional separations. With increases in scanning

References (140)

  • P. Davidsson et al.

    Peptide mapping of proteins in cerebrospinal fluid utilizing a rapid preparative two-dimensional electrophoretic procedure and matrix-assisted laser desorption/ionization mass spectrometry

    Biochim. Biophys. Acta

    (1999)
  • D. Wessel et al.

    A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids

    Anal. Biochem.

    (1984)
  • H. Schagger et al.

    Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa

    Anal. Biochem.

    (1987)
  • S. Hjerten

    Free zone electrophoresis

    Chromatogr. Rev.

    (1967)
  • J.W. Jorgenson et al.

    High-resolution separations based on electrophoresis and electroosmosis

    J. Chromatogr.

    (1981)
  • R. Haselberg et al.

    Capillary electrophoresis-mass spectrometry for the analysis of intact proteins

    J. Chromatogr. A

    (2007)
  • S. Hjerten et al.

    Adaptation of the equipment for high-performance electrophoresis to isoelectric-focusing

    J. Chromatogr.

    (1985)
  • J.D. Tipton et al.

    Analysis of intact protein isoforms by mass spectrometry

    J. Biol. Chem.

    (2011)
  • E.H. Seeley et al.

    MALDI imaging mass spectrometry of human tissue: method challenges and clinical perspectives

    Trends Biotechnol.

    (2011)
  • M.B. Comisarow et al.

    Fourier transform ion cyclotron resonance spectroscopy

    Chem. Phys. Lett.

    (1974)
  • M.B. Comisarow et al.

    Frequency-sweep fourier transform ion cyclotron resonance spectroscopy

    Chem. Phys. Lett.

    (1974)
  • S.C. Beu et al.

    Improved Fourier-transform ion-cyclotron-resonance mass spectrometry of large biomolecules

    J. Am. Soc. Mass Spectrom.

    (1993)
  • M.E. Belov et al.

    Electrospray ionization-fourier transform ion cyclotron mass spectrometry using ion preselection and external accumulation for ultrahigh sensitivity

    J. Am. Soc. Mass Spectrom.

    (2001)
  • M. Senko et al.

    External accumulation of ions for enhanced electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

    J. Am. Soc. Mass Spectrom.

    (1997)
  • B. Wilcox et al.

    Improved ion extraction from a linear octopole ion trap: SIMION analysis and experimental demonstration

    J. Am. Soc. Mass Spectrom.

    (2002)
  • S.M. Patrie et al.

    Construction of a hybrid quadrupole/Fourier transform ion cyclotron resonance mass spectrometer for versatile MS/MS above 10 kDa

    J. Am. Soc. Mass Spectrom.

    (2004)
  • M.J. Roth et al.

    Precise and parallel characterization of coding polymorphisms, alternative splicing, and modifications in human proteins by mass spectrometry

    Mol. Cell. Proteomics

    (2005)
  • C.M. Ryan et al.

    Post-translational modifications of integral membrane proteins resolved by top-down Fourier transform mass spectrometry with collisionally activated dissociation

    Mol. Cell. Proteomics

    (2010)
  • J.T. Ferguson et al.

    Top-down proteomics reveals novel protein forms expressed in Methanosarcina acetivorans

    J. Am. Soc. Mass Spectrom.

    (2009)
  • R. Aebersold et al.

    Mass spectrometry-based proteomics

    Nature

    (2003)
  • N.L. Kelleher

    Top-down proteomics

    Anal. Chem.

    (2004)
  • B.T. Chait

    Mass spectrometry: Bottom-up or top-down?

    Science

    (2006)
  • L.M. Smith et al.

    Proteoform: a single term describing protein complexity

    Nat. Methods

    (2013)
  • N. Siuti et al.

    Gene-specific characterization of human histone H2B by electron capture dissociation

    J. Proteome Res.

    (2006)
  • A. Resemann et al.

    Top-down de Novo protein sequencing of a 13.6 kDa camelid single heavy chain antibody by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry

    Anal. Chem.

    (2010)
  • J. Zhang et al.

    Top-down quantitative proteomics identified phosphorylation of cardiac troponin I as a candidate biomarker for chronic heart failure

    J. Proteome Res.

    (2011)
  • N.P. Barrera et al.

    Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions

    Nat. Methods

    (2009)
  • J. Erales et al.

    Mapping of a copper-binding site on the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis

    Biochem. J.

    (2009)
  • J.F. Kellie et al.

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

    Mol. Biosyst.

    (2010)
  • C.M. Whitehouse et al.

    Electrospray interface for liquid chromatographs and mass spectrometers

    Anal. Chem.

    (1985)
  • J.R. Johnson et al.

    Fourier-transform mass spectrometry for automated fragmentation and identification of 5–20 kDa proteins in mixtures

    Electrophoresis

    (2002)
  • F.Y. Meng et al.

    Processing complex mixtures of intact proteins for direct analysis by mass spectrometry

    Anal. Chem.

    (2002)
  • B.E. Chong et al.

    Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method

    Rapid Commun. Mass Spectrom.

    (2001)
  • D.B. Wall et al.

    Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry

    Anal. Chem.

    (2000)
  • D.B. Wall et al.

    Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line

    Rapid Commun. Mass Spectrom.

    (2001)
  • M.J. Roth et al.

    Sensitive and reproducible intact mass analysis of complex protein mixtures with superficially porous capillary reversed-phase liquid chromatography mass spectrometry

    Anal. Chem.

    (2011)
  • C.D. Wenger et al.

    Versatile online–offline engine for automated acquisition of high-resolution tandem mass spectra

    Anal. Chem.

    (2008)
  • A. Vellaichamy et al.

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

    Anal. Chem.

    (2010)
  • S.W. Lee et al.

    Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

    Proc. Natl. Acad. Sci. U.S.A.

    (2002)
  • J. Carroll et al.

    Definition of the mitochondrial proteome by measurement of molecular masses of membrane proteins

    Proc. Natl. Acad. Sci. U.S.A.

    (2006)
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