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Sep 1, 2011 (Vol. 31, No. 15)

Mass Spec Dominating Protein Profiling

Latest Enhancements Have Allowed Technique to Become a Rising Star in Proteomics Studies

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    Waters is investigating alternative methodologies for molecular characterization including ion-mobility spectrometry combined with mass spec to distinguish isobaric metabolites.

    Mass spectrometry has emerged as the leading technology for protein characterization. New instrumentation is more versatile and user friendly, expanding the application of mass spec technology to areas that were previously beyond its scope.

    A range of new offerings in mass spec instrumentation and their application to protein profiling were discussed at Waters' recent “Mass Spectrometry” conference.

    “In the field of proteomics today, the current methods of choice for molecular characterization are liquid chromatography combined with mass spectrometry, tandem mass spec, and nuclear magnetic resonance mass spectrometry,” said James Langridge, Ph.D., director of proteomics at Waters.

    “However, none of these approaches provides an unambiguous profile of target molecules. Therefore our team has investigated ion-mobility spectrometry combined with mass spec to distinguish isobaric—those having the same molecular weight—metabolites.”

    As Dr. Langridge pointed out, a particular atomic group may be positioned on a ring structure in one of several positions that are indistinguishable by mass spectrometry. However, with ion-mobility separation, the shape of the molecule enters into the equation and the two isobars can be separated into two distinct peaks.

    The T-wave ion-mobility separation is performed by the Waters Synapt high-definition MS system, which combines ion-mobility-based measurements and separations with quadrupole time of flight MS.

    The Waters system is built around the concept of a tri-partite device, the Triwave, in which the gaseous ions are accumulated in a trap. They are then moved to the ion-mobility separation segment of the Triwave and finally transferred to the TOF segment of the machine where they are subjected to mass analysis.

    The Synapt system can perform a number of operations that are important in proteomics studies, according to Dr. Langridge. These include analysis of intact proteins and protein complexes, enhanced protein sequencing, and improved protein identification.

    “I believe that the Triwave technology provides a number of key benefits, including the formation of first- and second-generation product ions from a precursor in a single experiments and the ability to perform a wide spectrum of tasks, such as characterization of post-translational modifications, maximization of sample information content, and a high-resolution analysis of small molecules,” Dr. Langridge concluded.

  • Interrogating the Membrane Proteome

    As quantitative investigations flesh out the texture of the proteome, it has become evident that around one-third is composed of membrane proteins. However, membrane proteins tend be to be large and unwieldy, difficult to purify while still retaining functionality, and hard to crystallize.

    For these reasons they are underrepresented in proteomics studies, and investigators have often ignored them despite their pivotal importance in cell function.

    This has changed in recent years, explained Kathryn Lilley, Ph.D., assistant director of research at the Cambridge Centre for Proteomics and the department of biochemistry at the University of Cambridge. She noted that membrane proteins are of special interest to the pharma industry, given the importance of receptors as targets of specific drugs.

    There are still challenges to be considered, however. “Questions raised when targeting membrane proteins include abundance, solubility, and suitability for standard proteomics workflow operations,” stated Dr. Lilley.

    She and her co-workers addressed these issues through the development of proteomics tools to define the expression of resistance nodulation division efflux pumps—which are low abundance, membrane-bound proteins—in Pseudomonas aeruginosa. Mutations in these proteins are a frequent cause of multiple drug resistance.

    Absolute quantification of the amount of protein requires the use of a marker peptide as a standard. Dr. Lilley chose enolase and calibrated the absolute abundance based on the performance of its top three scoring peptides. This approach is extremely flexible, she said, and applies over several orders of magnitude.

    Dr. Lilley discussed a second example of her approach to membrane protein characterization known as selected reaction monitoring, a complementary proteomic procedure based on the targeted analysis of a set of predetermined proteins and peptides.

    Selected peptides, based on their mass to charge ratio, are fragmented in the collision cell of a triple quadrupole mass spectrometer. The detected fragment ions, referred to as transitions, are used to construct a specific and highly sensitive assay for the detection of a particular peptide in a sample.

    This approach was also combined with global mass spectrometry-based protein localization studies in experiments aimed at localizing particular membrane proteins to the plant Golgi apparatus.

    “We found that a global membrane proteome analysis is more achievable by a combination of a variety of different mass spectrometry approaches,” Dr. Lilley said. “We have employed quantification of the membrane protein targets, identification of their binding partners, and a focus on the subcellular location of possible candidates for development of workable hypotheses.”


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