Tandem mass spectrometry, also known as MS/MS or MS2, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.
Peptide mass fingerprinting (PMF) is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer such as MALDI-TOF or ESI-TOF. The method was developed in 1993 by several groups independently. The peptide masses are compared to either a database containing known protein sequences or even the genome. This is achieved by using computer programs that translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides, and calculate the absolute masses of the peptides from each protein. They then compare the masses of the peptides of the unknown protein to the theoretical peptide masses of each protein encoded in the genome. The results are statistically analyzed to find the best match.
The advantage of this method is that only the masses of the peptides have to be known. Time-consuming de novo peptide sequencing is then unnecessary. A disadvantage is that the protein sequence has to be present in the database of interest. Additionally most PMF algorithms assume that the peptides come from a single protein. The presence of a mixture can significantly complicate the analysis and potentially compromise the results. Typical for the PMF based protein identification is the requirement for an isolated protein. Mixtures exceeding a number of 2-3 proteins typically require the additional use of MS/MS based protein identification to achieve sufficient specificity of identification (6). Therefore, the typical PMF samples are isolated proteins from two-dimensional gel electrophoresis or isolated SDS-PAGE bands. Additional analyses by MS/MS can either be direct, e.g., MALDI-TOF/TOF analysis or downstream nanoLC-ESI-MS/MS analysis of gel spot eluates.
In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation. It has been applied to the analysis of biomolecules and large organic molecules, which tend to be fragile and fragment when ionized by more conventional ionization methods. It is similar in character to electrospray ionization (ESI) in that both techniques are relatively soft ways of obtaining ions of large molecules in the gas phase, though MALDI typically produces far fewer multi-charged ions.
The Trans-Proteomic Pipeline (TPP) is an open-source data analysis software for proteomics developed at the Institute for Systems Biology (ISB) by the Ruedi Aebersold group under the Seattle Proteome Center. The TPP includes PeptideProphet, ProteinProphet, ASAPRatio, XPRESS and Libra.
A peptide sequence tag is a piece of information about a peptide obtained by tandem mass spectrometry that can be used to identify this peptide in a protein database.
Mascot is a software search engine that uses mass spectrometry data to identify proteins from peptide sequence databases. Mascot is widely used by research facilities around the world. Mascot uses a probabilistic scoring algorithm for protein identification that was adapted from the MOWSE algorithm. Mascot is freely available to use on the website of Matrix Science. A License is required for in-house use where more features can be incorporated.
PEAKS is a proteomics software program for tandem mass spectrometry designed for peptide sequencing, protein identification and quantification.
Electron-transfer dissociation (ETD) is a method of fragmenting multiply-charged gaseous macromolecules in a mass spectrometer between the stages of tandem mass spectrometry (MS/MS). Similar to electron-capture dissociation, ETD induces fragmentation of large, multiply-charged cations by transferring electrons to them. ETD is used extensively with polymers and biological molecules such as proteins and peptides for sequence analysis. Transferring an electron causes peptide backbone cleavage into c- and z-ions while leaving labile post translational modifications (PTM) intact. The technique only works well for higher charge state peptide or polymer ions (z>2). However, relative to collision-induced dissociation (CID), ETD is advantageous for the fragmentation of longer peptides or even entire proteins. This makes the technique important for top-down proteomics.The method was developed by Hunt and coworkers at the University of Virginia.
MALDI imaging mass spectrometry (MALDI-IMS) is the use of matrix-assisted laser desorption ionization as a mass spectrometry imaging technique in which the sample, often a thin tissue section, is moved in two dimensions while the mass spectrum is recorded. Advantages, like measuring the distribution of a large amount of analytes at one time without destroying the sample, make it a useful method in tissue-based study.
Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. It can also be used to localize proteins to the various organelles, and determine the interactions between different proteins as well as with membrane lipids.
Shotgun proteomics refers to the use of bottom-up proteomics techniques in identifying proteins in complex mixtures using a combination of high performance liquid chromatography combined with mass spectrometry. The name is derived from shotgun sequencing of DNA which is itself named after the rapidly expanding, quasi-random firing pattern of a shotgun. The most common method of shotgun proteomics starts with the proteins in the mixture being digested and the resulting peptides are separated by liquid chromatography. Tandem mass spectrometry is then used to identify the peptides.
Top-down proteomics is a method of protein identification that uses an ion trapping mass spectrometer to store an isolated protein ion for mass measurement and tandem mass spectrometry analysis. Top-down proteomics is capable of identifying and quantitating unique proteoforms through the analysis of intact proteins. The name is derived from the similar approach to DNA sequencing. Proteins are typically ionized by electrospray ionization and trapped in a Fourier transform ion cyclotron resonance, quadrupole ion trap or Orbitrap mass spectrometer. Fragmentation for tandem mass spectrometry is accomplished by electron-capture dissociation or electron-transfer dissociation. Effective fractionation is critical for sample handling before mass-spectrometry-based proteomics. Proteome analysis routinely involves digesting intact proteins followed by inferred protein identification using mass spectrometry. Top-down proteomics interrogates protein structure through measurement of an intact mass followed by direct ion dissociation in the gas phase.
Isobaric tags for relative and absolute quantitation (iTRAQ) is an isobaric labeling method used in quantitative proteomics by tandem mass spectrometry to determine the amount of proteins from different sources in a single experiment. It uses stable isotope labeled molecules that can be covalent bonded to the N-terminus and side chain amines of proteins.
A hybrid mass spectrometer is a device for tandem mass spectrometry that consists of a combination of two or more m/z separation devices of different types.
Selected reaction monitoring (SRM) is a method used in tandem mass spectrometry in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for detection.
Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics. Peptides or proteins are labeled with various chemical groups that are identical masses (isobaric), but vary in terms of distribution of heavy isotopes around their structure. These tags, commonly referred to as tandem mass tags, are designed so that the mass tag is cleaved at a specific linker region upon high-energy CID (HCD) during tandem mass spectrometry yielding reporter ions of different masses. The most common isobaric tags are amine-reactive tags. However, tags that react with cysteine residues and carbonyl groups have also been described. These amine-reactive groups go through N-hydroxysuccinimide (NHS) reactions, which are based around three types of functional groups. Isobaric labeling methods include tandem mass tags (TMT), isobaric tags for absolute and relative quantification (iTRAQ), mass differential tags for absolute and relative quantification, and dimethyl labeling. TMTs and iTRAQ methods are most common and developed of these methods. Tandem mass tags have a mass reporter region, a cleavable linker region, a mass normalization region, and a protein reactive group and have the same total mass.
In bio-informatics, a peptide-mass fingerprint or peptide-mass map is a mass spectrum of a mixture of peptides that comes from a digested protein being analyzed. The mass spectrum serves as a fingerprint in the sense that it is a pattern that can serve to identify the protein. The method for forming a peptide-mass fingerprint, developed in 1993, consists of isolating a protein, breaking it down into individual peptides, and determining the masses of the peptides through some form of mass spectrometry. Once formed, a peptide-mass fingerprint can be used to search in databases for related protein or even genomic sequences, making it a powerful tool for annotation of protein-coding genes.
In mass spectrometry, de novo peptide sequencing is the method in which a peptide amino acid sequence is determined from tandem mass spectrometry.
