Mass Spectrometry

Eric J. Sundberg, Ph.D., Scientist
Renne Lu, Ph.D., Senior Scientist

A mass spectrometer is an instrument for determining the mass and charge of a molecule. During the past decade, mass spectrometry has been increasingly applied to biological samples thanks to advances in ionization, fragmentation and detection techniques. BBRI has two mass spectrometers: (1) an electrospray ionization (ESI) linear ion-trap tandem mass spectrometer equipped with electron-transfer dissociation (ETD) fragmentation device; and (2) a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer.

BBRI has recently installed a state-of-the-art Thermo LTQ-XL ESI linear ion trap mass spectrometer equipped with a dual-pump Thermo Surveyor high pressure liquid chromatography (HPLC) unit. The LTQ-XL is capable of detecting all of the precursor ions (i.e., enzymatically-digested peptides) entering the instrument via the electrospray unit at a given time, trapping the most abundant ion, fragmenting that ion and detecting the fragments in a secondary mass spectrometer. The mass spectrum of the ions resulting from the fragmented precursor ion can then be matched to a database of mass spectra resulting from an in silico enzymatic digest of the relevant protein population (from a single protein to an entire proteome) in order to identify its amino acid sequence. Up to eight such iterative rounds of precursor ion detection, trapping, fragmentation and secondary mass spectrometric analysis can be performed per second by this instrument on successively less abundant tryptic peptides. Coupled with the resolving power of two-dimensional liquid chromatography, in which the orthogonal separation techniques of strong cation exchange and reverse phase separation of peptides are employed, it is possible to identify tens of thousands of peptides in a sample, and subsequently the thousands of proteins from which they came.

Our instrument is equipped not only with the standard fragmentation method, collision-induced dissociation (CID), but also with a novel and distinct fragmentation method, electron transfer dissociation (ETD). Fragmentation of precursor ions by CID and ETD results in the peptide backbone being cleaved at distinct sites, yielding unique ion fragments (b and y ions versus c and z ions, respectively). ETD is especially useful for the analysis of post-translational modifications since the fragmentation of the precursor ion is such that most modifications (e.g., phosphorylation) remain covalently attached to resulting fragment ions and provide a readily interpretable mass signature. Conversely, chemical groups attached to proteins via most post-translational modification events are normally the most labile portions of precursor ions CID fragmentation. This has two major effects: the release of the modifying group exhibits a particularly strong signal in the mass spectrometer, which results in suppression of the signals from the amino acid components of the peptide, making it difficult to determine the sequence of the peptide; and, because the modifying group is released unattached to amino acid component of the peptide it is often difficult to determine to which residue within the peptide this group had been attached originally, especially when more than one possible modification sites exist within the peptide (i.e., more than one Ser, Thr, or Tyr residue).

MALDI techniques facilitate vaporization and ionization of biological samples from a solid state into the gas phase.  When a molecule carrying a positive or negative charge travels through a long tube in an electric field, its velocity depends on its charge and its mass: the smaller a mass per charge is, the faster the molecule will travel. If the time required to reach the detector at the other end of the tube is measured, the mass of the molecule can be calculated. This type of instrument, which BBRI purchased using grants awarded by NIH and NSF, is called MALDI-TOF mass spectrometer.

The precision offered by MALDI-TOF mass spectrometry allows us to detect minute changes in the mass of a molecule. Mass spectrometry can therefore be used in a wide range of applications in research laboratories or pharmaceutical companies.  Common applications include quality control of synthetic peptides and drugs, verification of proteins and protein fragments generated by recombinant DNA techniques, and identification of phosphorylation and other post-translational modifications of proteins, which are often critical for the regulation and functions of proteins.

The ability of the MALDI mass spectrometer to analyze a complex mixture and its superb sensitivity make this instrument one of the most powerful tools in proteomics during the current post-genomic era.  An unknown protein may be cleaved into smaller fragments by an enzyme or chemically and the masses of the fragments in the mixture can be determined by mass spectrometry.  Upon comparison of the results with information in the genomic database, the original parent protein can be unambiguously identified.  This instrument also allows the identification of proteins associated with certain diseases, either up- or down-regulated, or binding partners of a molecule with known functions.