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MS

 

Mass Spectrometry

Mass spectrometry has a key role in the Center for Molecular and Cellular Systems identifying the proteins making up the protein complexes, or "machines of life," that are the focus of the Center. Mass spectrometry is a diverse family of instrumental techniques that have in common the measurement of molecular mass, and the study of reactions gas-phase ions. For a broader view of the capabilities and applications of mass spectrometry, visit the American Society for Mass Spectrometry web site.

Several distinct types of mass spectrometric measurements are used to identify proteins in the Center for Molecular and Cellular Systems. The goal of these measurements is the identification of proteins based on measurement of (a) molecular masses of proteins or proteolytic peptides, and (b) fragmentation patterns of proteins or proteolytic peptides obtained via tandem mass spectrometry. Mass spectrometric measurements of intact proteins are called "top-down," while measurements of proteolytic peptides are called "bottom-up." All of these measurements can be performed on either "workhorse" instruments or higher-performance mass spectrometers, depending on the desired accuracy and throughput.

"Bottom-up" tandem mass spectrometric analysis of the peptides resulting from proteolytic digests of proteins is a very high throughput method for identifying proteins. In the CMCS, we use quadrupole ion trap mass spectrometry to do these analyses. The tandem mass spectrum of a peptide provides partial amino acid sequence information about the peptide. Using a combination of commercial packages and software developed collaboratively in-house, this partial sequence information can be compared with known amino acid sequences in protein databases to yield protein identifications. To accommodate the very complex mixtures of peptides resulting from proteolytic digestion of a protein complex, each of the mass spectrometers is interfaced with high-performance liquid chromatography equipment to perform on-line separations. A versatile array of single- and multi-dimensional chromatographic separations, combined with tailored mass spectometry methods, allows us to accommodate proteolytic digests ranging in complexity from simple protein mixtures (e.g. affinity-isolated protein complexes) through full microbial cell proteomes.

The quadrupole ion trap mass spectrometers used for high-throughput protein identification are robust, but do not offer particularly high performance with regard to mass measurement accuracy and resolution. Higher-performance mass spectrometers, such as Fourier transform ion cyclotron resonance or hybrid quadrupole-time-of-flight mass spectrometers, offer more powerful measurements, at the expense of speed. At PNNL, an approach called the accurate mass and time (AMT) tag method has been developed. In this approach, measurements of peptide masses are obtained that are sufficiently accurate (in the parts-per-million range) to identify the peptides without the need for tandem mass spectrometry. At ORNL, "top-down" measurements of intact protein masses are performed using Fourier transform ion cyclotron resonance mass spectrometry. These measurements allow identification of modified versions of proteins that could be missed in a "bottom-up" approach that looks only at peptides.

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