Mass Spectrometry Applications for Proteomics

Click to view the PDF file: Proteomic Mass Spectrometry Equipment Courtesy of CiphergenCiphergen's SELDI process, a MALDI variant that includes a surface-based enrichment step Early in the twentieth century, scientists puzzled over the observation that certain elements that were otherwise physically indistinguishable from each other nevertheless exhibited different radioactive decay characteristics. These elements would ultimately come to be known as isotopes, but at the time this concept was

By | August 20, 2001

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Proteomic Mass Spectrometry Equipment

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Courtesy of Ciphergen

Ciphergen's SELDI process, a MALDI variant that includes a surface-based enrichment step

Early in the twentieth century, scientists puzzled over the observation that certain elements that were otherwise physically indistinguishable from each other nevertheless exhibited different radioactive decay characteristics. These elements would ultimately come to be known as isotopes, but at the time this concept was a mystery. Then, in 1912 J. J. Thomson designed a device in which positive rays generated by the ionization of a gas in a discharge tube were subjected to both magnetic and electric fields, and separated the two isotopes of neon, with masses of 20 and 22. Thus was born the first mass spectrometer, a device that, in conjunction with microarray analysis and two-dimensional gel electrophoresis (2DE), is now driving the proteomics revolution forward. Today, researchers use mass spectrometers to identify proteins, sequence peptides, identify posttranslational modifications, and characterize multi-protein complexes.

Proteomics, which is generally defined as the characterization of the complete protein complement of a cell, tissue, or organism (a proteome), has moved to the forefront of "big science" now that the complete genomes of several organisms--including man--have been sequenced.1 But why is proteomics important? Simply put, proteins define the function of cells, tissues, and even organisms. Every cell in the human body contains an equivalent set of genes, but not every cell expresses the same ones. Only B cells, for example, express immunoglobulins. In addition, each gene can give rise to multiple proteins, either through alternative splicing or post-translational modifications. Finally, there is no direct correlation between mRNA abundance and steady-state protein levels.2 For all of these reasons, the field of proteomics is booming.

Brian T. Chait is the director of the National Resource for Mass Spectrometric Analysis of Biological Macromolecules at Rockefeller University, one of several National Institutes of Health National Centers for Research Resources (NCRR) around the country. Chait describes proteomics as "a style of doing biology, where one tries to look at many things at the same time, and gather lots of information, rather than gathering it one protein at a time." He makes the analogy of studying an automobile: It is very difficult to understand how a car works if it is taken entirely apart and studied piece by piece. Similarly, "if you dissect out a particular protein, and look at it in detail, you could miss the point of it. You have to look at it in context, in vivo, in its full complexity of interactions," Chait says.

Proteomics began over 25 years ago with the development of 2DE.3 Early attempts to catalog the individual protein "spots" found on these gels, however, proved difficult owing to problems with gel-to-gel reproducibility and protein sequencing. Scientists needed a protein identification method that, unlike the standard Edman degradation protocol, could analyze the tiny amounts of sample found on these gels. Mass spectrometry (MS) fit this bill very nicely. Unfortunately, however, MS analyzes ionized samples in gaseous form, and there was no way to get peptides into the gas phase. Recent advances in ionization technology have overcome this problem, and it is now possible to sequence and analyze femtomole (10-15 moles) quantities of peptide.

Ionization of Biological Samples

Mass spectrometers generally couple three devices: an ionization device, a mass analyzer, and a detector. The most common ionization techniques used in biology are matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). These two "soft ionization" methods induce very little sample degradation during the ionization process. MALDI is most commonly used to produce a preliminary scan of the peptide components released from a sample by proteolytic digestion, such as the proteins present in an acrylamide gel slice. It is a solid phase technique in that researchers mix a peptide sample with a huge excess of matrix material, usually either a-cyano-4-hydroxycinnamic acid or dihydrobenzoic acid, and precipitate the mixture on a plate by drying. These matrices are chosen because they absorb energy at the wavelength of the laser, usually a nitrogen laser at 337 nm. As the matrix absorbs the laser energy, it emits heat, causing vaporization and ionization of the sample, generally by protonation of the peptide to a charge of +1.

Whereas MALDI is a solid-state and pulsed process, ESI is a liquid phase and usually continuous technology. Scientists ionize peptide samples via ESI by forcing a liquid sample through a small-bored capillary tip in the presence of an electrical charge, producing a fine mist of ionized droplets. As these droplets dry, the peptides within the drop concentrate until electrostatic repulsion forces the droplet apart, producing a cloud of ionized peptides with a charge of at least +2. Because it starts with peptides in solution, ESI is compatible with both high-pressure liquid chromatography (HPLC) and capillary electrophoresis, both of which may be used to concentrate and purify individual peptides prior to mass analysis.

Michael Gross, director of the Washington University Center for Biomedical and Bioorganic Mass Spectrometry, another NCRR, observes that the development of MALDI and ESI was "evolutionary. But their impact," he says, "is perhaps revolutionary." He notes, for example, that before there was MALDI, mass spectrometrists had fast atom bombardment ionization, a matrix-based technique that uses atomic collisions to ionize the sample, instead of a laser. Similarly, the concept of laser desorption has been known for over 20 years. The union of these two techniques in MALDI, however, vastly enhanced the ability of researchers to analyze biological samples.

Mass Analysis

Once a sample has been ionized, it must be mass analyzed. MS instruments, despite their names, do not actually provide a mass value. Instead, they report the mass-to-charge (m/z) ratio. If the charge of the ion is known, the mass can be calculated. The most commonly used mass analyzers for protein biochemistry applications are time-of-flight (TOF), triple-quadrupole, quadrupole-TOF, and ion trap instruments.

Courtesy of Micromass-Waters

Schematic diagram of Micromass's Q-tof Ultima API, a tandem mass spectrometer using an ESI source and quadrupole/hexapole/TOF tandem mass analyzer configuration.

The TOF analyzer is conceptually the simplest spectrometer.4 In a TOF MS, each ion has the same initial kinetic energy, but a speed that varies with mass. A reflector (called a "reflectron"), used to compensate for minor differences in initial kinetic energy, redirects the ions back towards the detector, focusing the beam at the same time. A detector then records each ion that strikes it. Since ions have a mass-dependent velocity, the machine can determine mass by each ion's time-of-flight. TOF instruments can theoretically detect all ions that are produced in an ionization event, and are therefore described as having a "multi-channel advantage." They are also highly compatible with fast, pulsed ionization methods such as MALDI.

Quadrupole analyzers consist of four parallel rods arranged as a square, across which an oscillating electrical field is applied. Ions travel through the empty space in the center of the square, but only ions with a specific m/z ratio can reach the detector, based upon the field generated by the charge applied to the rods.4 By varying the electrical field, different ions can be analyzed. "Quad" machines are typically coupled to ESI devices, often in tandem configurations (MS/MS). For example, the "triple-quad" configuration consists of three quadrupole MS analyzers. Tandem MS machines generally have three functions: ion selection, ion fragmentation, and mass analysis. Researchers apply the sample to the first machine, which filters the sample components for a specific ion based on its m/z ratio. This ion is then sent to the second chamber, which functions as a "collision chamber," fragmenting the ion into a series of product ions by collision with an introduced neutral gas. Finally, the product ions are mass analyzed in the third analyzer. Because quadrupole instruments are not multi-channel (that is, because they cannot analyze every incoming ion simultaneously), the state-of-the-art MS machines use a TOF instrument in the third position instead of another quadrupole (Q-TOF).

Ion trap apparatuses are single analyzers that function like a tandem machine in time, rather than in space. Introduced peptide samples are filtered during the injection process so that only the desired peptide accumulates. Then they are fragmented into product ions inside the device, and then finally mass analyzed by controlled ejection of ions towards a detector over a mass range. Thus, like a TOF instrument, the ion trap has a multi-channel advantage.4 Ion traps are particularly useful in structural studies, because they allow successive collision reactions to be performed and analyzed.

A complete MS instrument contains an ionization source, a mass analyzer, and a detector. Typical configurations for biological applications are MALDI-TOF and ESI coupled to an ion trap, triple-quad, or Q-TOF. However, Gross notes that as long as the MS is capable of receiving and processing the ions any ionization method can be coupled to any MS configuration. "It's as if you had column A and column B in a Chinese restaurant," he explains. Incompatibilities arise, however, if the analysis speed of the MS instrument is too slow for the ionization method. Thus, it is difficult to couple a MALDI source to a triple-quadrupole instrument, because the MALDI laser fires at a frequency in the millisecond range, while the mass analyzer might require a few seconds to complete its scan.

Mass Spectrometry Applications

The number of proteomic applications of MS is constantly growing as researchers develop more improved instruments as well as clever ways to couple them. Four common uses of MS are discussed here.

Protein Identification: The most common biological application of MS is the identification of an unknown protein or proteins in a sample. The input protein sample might be a single spot from a 2DE experiment or a band purified from an SDS-PAGE 1D gel. Researchers excise the region of gel containing the protein of interest, digest the sample "in-gel" with trypsin (which cleaves proteins on the C-terminal side of either lysine or arginine), and elute the peptides from the acrylamide. These peptides are then subjected to MALDI-TOF, producing a series of peaks, each describing the molecular mass of a single peptide in the mixture.

Because it is possible to know exactly which tryptic fragments any given protein can generate, the set of peptide masses resulting from this type of experiment can be used as a "fingerprint" of the parent protein--that is, only one of the proteins from a particular organism could produce the observed series of peptides. Researchers then query sequence databases looking for matches. Obviously, the confidence of a given match depends on the accuracy of the mass measurement. Huge sequence databases that enable the researcher to compare the observed peptide fingerprint with, for example, every possible human protein, amplify the power of this technique.

Researchers have imagined that the combination of 2DE with MS would be a proteomic panacea. However, recent work by Steven P. Gygi, Ruedi Aebersold, and colleagues casts doubt on the usefulness of this approach.5 These authors separated a crude yeast lysate via 2DE, and examined 50 protein spots within a 4-cm2 region. Their observations exposed a number of shortcomings to the 2DE-MS approach, including comigration of different proteins to a single location and differential migration of the product of a single gene to multiple locations. Most importantly, these researchers found that low-abundance proteins went undetected, despite the fact that such proteins represent the majority of proteins encoded by the yeast genome, and were predicted to be present within the 4-cm2 area examined. They conclude that 2DE-MS approaches will not prove to be an effective proteomic tool without enrichment of low-abundance proteins prior to MS analysis.

Peptide Sequencing: If the set of peptides resulting from the MALDI-TOF analysis is not specific enough to conclusively identify a single protein, then the next step is peptide sequencing. This is generally accomplished using an ESI source coupled to a tandem MS instrument (either an ion trap, Q-TOF, or triple-quadrupole analyzer). An HPLC or CE instrument will be coupled on-line as well, to enable prefractionation of the peptide mixture. Researchers introduce the desired peptide into the machine, fragment it along the peptide backbone via collision-induced dissociation, and then mass analyze the resulting fragments. Two or more series of fragments are produced, depending on where the peptide bonds break. Fragments from one series will vary in mass from each other by the weight of a single amino acid, enabling the researcher to deduce the peptide sequence. For example, in a tryptic digestion, the C-terminal residue in the peptide chain is either lysine (145) or arginine (173). The mass of next larger fragment should then be the sum of 145 or 173, plus the mass of the next N-terminal residue. Ideally, peptide sequencing approaches will yield a "ladder" of peptide fragments, although in practice this is not always observed because only those fragments that include the charged ion can be analyzed.4

Identification of Posttranslational Modifications: Covalent modifications of peptides can also be detected via MS. Since the addition of phosphate groups increases the peptide mass by 80 Daltons, it is possible to determine whether a given peptide is phosphorylated by comparison of untreated and phosphatase-treated peptides via MALDI-TOF.6 Once a phosphorylated peptide has been identified, it can be sequenced using tandem MS. Jun Qin, assistant professor of biochemistry at the Baylor College of Medicine in Houston, Texas, has coauthored several papers that use this technique. He recently helped identify phosphorylated residues in both NFAT1 and Brca1, using MALDI-TOF followed by capillary liquid chromatography coupled to an ESI/ion trap-tandem MS device.7,8Glycosylation9 and sulfation sites can also be characterized by MS.

Characterization of Multi-Protein Complexes: Scientists can dissect multi-protein complexes through MS as well. Generally one of the components of the mixture is tagged, so that the complex can be isolated via affinity chromatography. Scientists then subject the resulting complex of proteins to MS analysis, either with or without prior fractionation. For example, Matthias Mann of the European Molecular Biology Laboratory in Heidelberg, Germany, and Kim Nasmyth of the Research Institute of Molecular Pathology in Vienna, Austria, collaborated on a project to identify components of the yeast anaphase-promoting complex.10 They tagged one member of the complex, Cdc16, with a c-Myc epitope, and immunoprecipitated the complex with an anti-Myc antibody. They then resolved the resulting protein products on a gel, purified them, and analyzed using nanoelectrospray tandem MS. Nanoelectrospray ESI technology operates at a very low flow rate, without pumps, and is particularly amenable to peptide sequencing applications.4

Odds and Ends

The application of MS to proteomics is considerably more extensive than this overview has made it appear. There are, for example, other types of mass analyzers, such as the Fourier transform-ion cyclotron resonance MS (FT-ICR). There are also other MS applications, such as structural analysis. A number of excellent reviews have been published on this subject (e.g., references 4, 11, 12), as well as a variety of textbooks, including Biological Mass Spectrometry, edited by Takekiyo Matsuo, Richard M. Caprioli, Michael Gross, and Yousuke Seyama,13 and Interpreting Protein Mass Spectra, by A. Peter Snyder.14

As has been the case with so much of "big science," the combination of sequence information, improved instrumentation, and increased affordability, is making protein biochemistry easier than ever. Yet, despite recent advances, the MS proteomics revolution has only just begun. The challenge for researchers now is to find ways to optimize sample handling procedures and pre-analysis fractionations so that accurate and comprehensive "whole proteome" studies can be performed reliably. As Gross explains, "Part of the excitement of the subject of MS is that it is still evolving and looking for ways of matching and mixing and exploiting these various tools, putting them together in combinations, to give you a yet more powerful biological tool than we have today." It is important to put MS in perspective. It is not an end, but a beginning. Says Rockefeller's Chait: "It is an enabling tool, and it's not magic. But tools are terrific, because they let you do all kinds of things, and open up all kinds of windows."

Jeffrey M. Perkel can be contacted at
1.E. Russo, "Behind the sequence," The Scientist, 15[5]:1, March 5, 2001.

2.S. Gygi et al., "Correlation between protein and mRNA abundance in yeast," Molecular and Cellular Biology, 19:1720-30, 1999.

3.H.E. Sussman, "2-D glasses," The Scientist, 15[7]:23, April 2, 2001.

4.M. Mann et al., "Analysis of proteins and proteomes by mass spectrometry," Annual Review of Biochemistry, 70:437-73, 2001.

5.S.P. Gygi et al., "Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology," Proceedings of the National Academy of Sciences, 97[17]:9390-5, Aug. 15, 2000.

6.X. Zhang et al., "Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis," Analytical Chemistry, 70:2050-9, 1999.

7.H. Okamura et al., "Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity," Molecular Cell, 6:539-50, Sept. 2000.

8.D. Cortez, "Requirement of ATM-dependent phosphorylation of Brca1 in the DNA damage response to double-strand breaks," Science, 286:1162-6, 1999.

9.A. Dell, H.R. Morris, "Glycoprotein structure determination by mass spectrometry," Science, 291:2351-6, March 23, 2001.

10.W. Zachariae et al., "Mass spectrometric analysis of the anaphase-promoting complex from yeast: Identification of a subunit related to cullins," Science, 279:1216-9, 1998.

11.J.R. Yates, "Mass spectrometry and the age of the proteome," Journal of Mass Spectrometry, 33:1-19, 1998.

12.A. Pandey, M. Mann, "Proteomics to study genes and genomes," Nature, 405:837-46, June 15, 2000.

13.T. Matsuo et al. (eds.), Biological Mass Spectrometry: Present and Future, New York: John Wiley & Sons, 1994.

14.A.P. Snyder, Interpreting Protein Mass Spectra: A Comprehensive Resource, New York: Oxford University Press, 2000.

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