Biochemical and Biophysical Research Communications
ReviewTop Down proteomics: Facts and perspectives
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
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