Protein mass spectrometry

From Wikipedia, the free encyclopedia
Jump to: navigation, search
A mass spectrometer used for high throughput protein analysis.

Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important emerging method for the characterization of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. This approach is referred to as "top-down" strategy of protein analysis. In the second, proteins are enzymatically digested into smaller peptides using a protease such as trypsin. Subsequently these peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry. Hence, this latter approach (also called "bottom-up" proteomics) uses identification at the peptide level to infer the existence of proteins.

Whole protein mass analysis is primarily conducted using either time-of-flight (TOF) MS, or Fourier transform ion cyclotron resonance (FT-ICR). These two types of instrument are preferable here because of their wide mass range, and in the case of FT-ICR, its high mass accuracy. Mass analysis of proteolytic peptides is a much more popular method of protein characterization, as cheaper instrument designs can be used for characterization. Additionally, sample preparation is easier once whole proteins have been digested into smaller peptide fragments. The most widely used instrument for peptide mass analysis are the MALDI time-of-flight instruments as they permit the acquisition of peptide mass fingerprints (PMFs) at high pace (1 PMF can be analyzed in approx. 10 sec). Multiple stage quadrupole-time-of-flight and the quadrupole ion trap also find use in this application.

History[edit]

The application of mass spectrometry to study proteins became popularized in the 1980's after the development of MALDI and ESI. These ionization techniques have played a significant role in the characterization of proteins. (MALDI) Matrix-assisted laser desorption ionization was coined in the late 80's by Franz Hillenkamp and Michael Karas.[1] Hillenkamp, Karas and their fellow researchers were able to ionize the amino acid alanine by mixing it with the amino acid tryptophan and irradiated with a pulse 266 nm laser. [2] Though important, the breakthrough did not come until 1987. In 1987, Koichi Tanaka used the "ultra fine metal plus liquid matrix method" and ionized biomolecules the size of 34,472 Da protein carboxypeptidase-A. [3]

In 1968, Malcolm Dole reported the first use of electrospray ionization with mass spectrometry. Around the same time MALDI became popularized, John Bennett Fenn was sited for the development of electrospray ionization. [4][5] Tanaka received the 2002 Nobel Prize in Chemistry along side John Fenn, and Kurt Wüthrich "for the development of methods for identification and structure analyses of biological macromolecules."[6] These ionization methods have made the study of protein applicable with mass spectrometry. Now protein mass spectrometry is a significant role in protein characterization.

Protein and peptide fractionation coupled with mass spectrometry[edit]

Mass spectrometry protocol

Proteins of interest are usually part of a complex mixture of multiple proteins and molecules, which co-exist in the biological medium. This presents two significant problems. First, the two ionization techniques used for large molecules only work well when the mixture contains roughly equal amounts of constituents, while in biological samples, different proteins tend to be present in widely differing amounts. If such a mixture is ionized using electrospray or MALDI, the more abundant species have a tendency to "drown" or suppress signals from less abundant ones. Second, mass spectrum from a complex mixture is very difficult to interpret due to the overwhelming number of mixture components. This is exacerbated by the fact that enzymatic digestion of a protein gives rise to a large number of peptide products.

In light of these problems, the methods of two-dimensional gel electrophoresis and high performance liquid chromatography are widely used for separation of proteins. The first method fractionates whole proteins two-dimensional gel electrophoresis. The first-dimension of 2D gel is isoelectric focusing (IEF). In this dimension, the protein is separated by its isoelectric point (pI) and the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This dimension separates the protein according to its molecular weight.[7] Once this step is completed in-gel digestion occurs. In some situations, it may be necessary to combine both of these techniques. Gel spots identified on a 2D Gel are usually attributable to one protein. If the identity of the protein is desired, usually the method of in-gel digestion is applied, where the protein spot of interest is excised, and digested proteolytically. The peptide masses resulting from the digestion can be determined by mass spectrometry using peptide mass fingerprinting. If this information does not allow unequivocal identification of the protein, its peptides can be subject to tandem mass spectrometry for de novo sequencing. Small changes in mass and charge can be detected with 2D-PAGE. The disadvantages with this technique are it’s small dynamic range compared to other methods, some proteins are still difficult to separate due to their acidity, basicity, hydrophobicity, and size (too large or too small).[8]

The second method, high performance liquid chromatography is used to fractionate peptides after enzymatic digestion. Characterization of protein mixtures using HPLC/MS is also called shotgun proteomics and MuDPIT (Multi-Dimensional Protein Identification Technology). A peptide mixture that results from digestion of a protein mixture is fractionated by one or two steps of liquid chromatography. The eluent from the chromatography stage can be either directly introduced to the mass spectrometer through electrospray ionization, or laid down on a series of small spots for later mass analysis using MALDI.

Protein identification[edit]

There are two main ways MS is used to identify proteins. Peptide mass fingerprinting uses the masses of proteolytic peptides as input to a search of a database of predicted masses that would arise from digestion of a list of known proteins. If a protein sequence in the reference list gives rise to a significant number of predicted masses that match the experimental values, there is some evidence that this protein was present in the original sample. Purification steps therefore limit the throughput of the peptide mass fingerprinting approach. Peptide mass fingerprinting can be achieved with the used for MS-MS.[9]

Chromatography trace and MS2 spectra of a peptide.

Tandem MS is a method for identifying proteins. Collision-induced dissociation is used in mainstream applications to generate a set of fragments from a specific peptide ion. The fragmentation process primarily gives rise to cleavage products that break along peptide bonds. Because of this simplicity in fragmentation, it is possible to use the observed fragment masses to match with a database of predicted masses for one of many given peptide sequences. Tandem MS of whole protein ions has been investigated recently using electron capture dissociation and has demonstrated extensive sequence information in principle but is not in common practice. This is sometimes referred to as the "top-down" approach in that it involves starting with the whole mass and then pulling it apart rather than starting with pieces (proteolytic fragments) and piecing the protein back together using de novo repeat detection (bottom-up).[9]

De novo (peptide) sequencing[edit]

De novo peptide sequencing for mass spectrometry is typically performed without prior knowledge of the amino acid sequence. It is the process of assigning amino acids from peptide fragment masses of a protein. De novo sequencing has proven successful for confirming and expanding upon results from database searches.

As de novo sequencing is based on mass and some amino acids have identical masses (e.g. leucine and isoleucine), accurate manual sequencing can be difficult. Therefore it may be necessary to utilize a sequence homology search application to work in tandem between a database search and de novo sequencing to address this inherent limitation.

Database searching has the advantage of quickly identifying sequences, provided they have already been documented in a database. Other inherent limitations of database searching include sequence modifications/mutations (some database searches do not adequately account for alterations to the 'documented' sequence, thus can miss valuable information), the unknown (if a sequence is not documented, it will not be found), false positives, and incomplete and corrupted data.[10]

An annotated peptide spectral library can also be used as a reference for protein/peptide identification. It offers the unique strength of reduced search space and increased specificity. The limitations include spectra not included in the library will not be identified, spectra collected from different types of mass spectrometers can have quite distinct features, and reference spectra in the library may contain noise peaks, which may lead to false positive identifications.[11] A number of different algorithmic approaches have been described to identify peptides and proteins from tandem mass spectrometry (MS/MS), peptide de novo sequencing and sequence tag-based searching.[12]

Protein quantitation[edit]

Quantitative Mass Spectrometry.

Several recent methods allow for the quantitation of proteins by mass spectrometry (quantitative proteomics). Typically, stable (e.g. non-radioactive) heavier isotopes of carbon (13C) or nitrogen (15N) are incorporated into one sample while the other one is labeled with corresponding light isotopes (e.g. 12C and 14N).[13] The two samples are mixed before the analysis. Peptides derived from the different samples can be distinguished due to their mass difference. The ratio of their peak intensities corresponds to the relative abundance ratio of the peptides (and proteins). The most popular methods for isotope labeling are SILAC (stable isotope labeling by amino acids in cell culture), trypsin-catalyzed 18O labeling, ICAT (isotope coded affinity tagging), iTRAQ (isobaric tags for relative and absolute quantitation).[14] “Semi-quantitative” mass spectrometry can be performed without labeling of samples.[15] Typically, this is done with MALDI analysis (in linear mode). The peak intensity, or the peak area, from individual molecules (typically proteins) is here correlated to the amount of protein in the sample. However, the individual signal depends on the primary structure of the protein, on the complexity of the sample, and on the settings of the instrument. Other types of "label-free" quantitative mass spectrometry, uses the spectral counts (or peptide counts) of digested proteins as a means for determining relative protein amounts.[10]

Protein structure[edit]

Characteristics indicative of the 3-dimensional structure of proteins can be probed with mass spectrometry in various ways.[16] By using chemical crosslinking to couple parts of the protein that are close in space, but far apart in sequence, information about the overall structure can be inferred. By following the exchange of amide protons with deuterium from the solvent, it is possible to probe the solvent accessibility of various parts of the protein.[17] Hydrogen-deuterium exchange mass spectrometry has been used to study proteins and their conformations for over 20 years. This type of protein structural analysis can be suitable for proteins that are challenging for other structural methods.[18] Another interesting avenue in protein structural studies is laser-induced covalent labeling. In this technique, solvent-exposed sites of the protein are modified by hydroxyl radicals. Its combination with rapid mixing has been used in protein folding studies.[19]

Practical applications[edit]

Biomarkers[edit]

The FDA defines a biomarker as, “A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”. It is hypothesized that mass spectrometry enables the discovery of candidates for biomarkers.

Proteogenomics[edit]

In what is now commonly referred to as proteogenomics, peptides identified with mass spectrometry are used for improving gene annotations (for example, gene start sites) and protein annotations. Parallel analysis of the genome and the proteome facilitates discovery of post-translational modifications and proteolytic events,[20] especially when comparing multiple species.[21]

References[edit]

  1. ^ Karas, M.; Bachmann, D.; Hillenkamp, F. (1985). "Influence of the Wavelength in High-Irradiance Ultraviolet Laser Desorption Mass Spectrometry of Organic Molecules". Analytical Chemistry 57 (14): 2935–9. doi:10.1021/ac00291a042. 
  2. ^ Karas, M.; Bachmann, D.; Hillenkamp, F. (1985). "Influence of the Wavelength in High-Irradiance Ultraviolet Laser Desorption Mass Spectrometry of Organic Molecules". Analytical Chemistry 57 (14): 2935–9. doi:10.1021/ac00291a042. 
  3. ^ Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. (1987). "Matrix-Assisted Ultraviolet Laser Desorption of Non-Volatile Compounds". International Journal of Mass Spectrometry and Ion Processes 78: 53–68. Bibcode:1987IJMSI..78...53K. doi:10.1016/0168-1176(87)87041-6. 
  4. ^ Dole M, Mack LL, Hines RL, Mobley RC, Ferguson LD, Alice MB (1968). "Molecular Beams of Macroions". Journal of Chemical Physics 49 (5): 2240–2249. Bibcode:1968JChPh..49.2240D. doi:10.1063/1.1670391. 
  5. ^ Birendra N. Pramanik; A.K. Ganguly; Michael L. Gross (28 February 2002). Applied Electrospray Mass Spectrometry: Practical Spectroscopy Series. CRC Press. pp. 4–. ISBN 978-0-8247-4419-9. 
  6. ^ "Press Release: The Nobel Prize in Chemistry 2002". The Nobel Foundation. 2002-10-09. Retrieved 2011-04-02. 
  7. ^ W. Wells, G. Wang and S. J. Baek (2006). "Comparative Study of Three Proteomic Quantitative Methods, DIGE, cICAT, and iTRAQ, Using 2D Gel- or LC−MALDI TOF/TOF". Journal of Proteome Research 5 (3): 651–658. doi:10.1021/pr050405o. PMID 16284939. 
  8. ^ Issaq, J. Haleem and T. D. Veenstra (2008). "Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE): advances and perspectives". BioTechniques 46 (5): 697–700. doi:10.2144/000112823. PMID 16284939. 
  9. ^ a b Trauger, A. Sunia and W. Webb and G. Siuzdak (2002). "Peptide and protein analysis with mass spectrometry". Spectroscopy 16 (1): 15–28. doi:10.1155/2002/320152. PMID 16284939. 
  10. ^ a b Bret, Cooper and J. Feng and W. Garrett (2010). "Relative, Label-free Protein Quantitation: Spectral Counting Error Statistics from Nine Replicate MudPIT Samples". Spectroscopy 21 (9): 1534–1546. doi:10.1016/j.jasms.2010.05.001. 
  11. ^ Augustin, Scalbert and L. Brennan and O. Fiehn (2009). "Mass-spectrometry-based metabolomics: limitations and recommendations for future progress with particular focus on nutrition research". NCBI 5 (4): 435–458. doi:10.1007/s11306-009-0168-0. 
  12. ^ P. Hernandez, M. Müller and R. D. Appel (2006). "Automated protein identification by tandem mass spectrometry: Issues and strategies". Mass Spectrometry Reviews 25 (2): 235–254. doi:10.1002/mas.20068. PMID 16284939. 
  13. ^ Snijders AP, de Vos MG, Wright PC (2005). "Novel approach for peptide quantitation and sequencing based on 15N and 13C metabolic labeling". J. Proteome Res. 4 (2): 578–85. doi:10.1021/pr0497733. PMID 15822937. 
  14. ^ M. Miyagi and K. C. S. Rao (2007). "Proteolytic 18O-labeling strategies for quantitative proteomics". Mass Spectrometry Reviews 26 (1): 121–136. doi:10.1002/mas.20116. PMID 17086517. 
  15. ^ Haqqani AS, Kelly JF, Stanimirovic DB (2008). "Quantitative protein profiling by mass spectrometry using label-free proteomics". Methods Mol. Biol. 439: 241–56. doi:10.1007/978-1-59745-188-8_17. PMID 18370108. 
  16. ^ Z. Zhang, D. L. Smith (1994). "Probing noncovalent structural features of proteins by mass spectrometry". Mass Spectrometry Reviews 13 (5–6): 411–429. doi:10.1002/mas.1280130503. 
  17. ^ T. E. Wales and J. R. Engen (2006). "Hydrogen exchange mass spectrometry for the analysis of protein dynamics". Mass Spectrometry Reviews 25 (1): 158–170. doi:10.1002/mas.20064. PMID 16208684. 
  18. ^ R.E Iacob and J. R. Engen (2012). "Hydrogen Exchange Mass Spectrometry: Are We Out of the Quicksand?". Mass Spectrometry Reviews 23 (6): 1003–1010. doi:10.1007/s13361-012-0377-z. PMID 22476891. 
  19. ^ B. B. Stocks and L. Konermann (2009). "Structural Characterization of Short-Lived Protein Unfolding Intermediates by Laser-Induced Oxidative Labeling and Mass Spectrometry". Anal. Chem. 81 (1): 20–27. doi:10.1021/ac801888h. 
  20. ^ Gupta N., Tanner S., Jaitly N., Adkins J.N., Lipton M., Edwards R., Romine M., Osterman A., Bafna V., Smith R.D., et al. Whole proteome analysis of post-translational modifications: Applications of mass-spectrometry for proteogenomic annotation. Genome Res. 2007;17:1362–1377.
  21. ^ Gupta N., Benhamida J., Bhargava V., Goodman D., Kain E., Kerman I., Nguyen N., Ollikainen N., Rodriguez J., Wang J., et al. Comparative proteogenomics: Combining mass spectrometry and comparative genomics to analyze multiple genomes. Genome Res. 2008;18:1133–1142.