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The Proteomics Facility has acquired and installed several instruments for micro-sequencing, structural analysis, and quantitative studies of proteins. Facility staff are available to assist in the characterization of both targeted protein complexes and trace components in lysates and sera.
Unbiased protein identification relies upon data-directed MS/MS structural investigations of digest peptides. Generally, the peptides are resolved by an organic gradient coupled to electrospray ionization (ESI). Decisions made in real time determine which peptides are interrogated for sequence assignment. Separations may also be conducted off-line, however, with robotic fraction collection directly to 384 micro well target plates. These are analyzed later by matrix assisted laser desorption ionization (MALDI). The plates can be stored and reexamined for up to three weeks. MALDI also enables robust detection of many biomolecules such as oligonucleotides, lipids, and glycoproteins.
The facility's ETD capability is a powerful tool for qualitative analysis that moves the bar beyond simple protein identification. ETD provides more uniform sequence coverage for peptiedes of 3+ and higher charge state, so it is complementary to CID. However, ETD-activated ions cleave to yield sequence specific fragments more rapidly than they incur loss of PTM or rearrangement (even H/D scrambling). Hence, the precise residues bearing characteristic mass modifications may be identified more routinely with ETD than with other activation techniques.
Preliminary stages of biomarker discovery often require unbiased approaches to comparative proteomics. The core supports SILAC, iTRAQ or chemical labeling protocols that are often combined with 2 dimensional separations prior to MS/MS. Regulated protein expression is deduced by the distinct ratios of constituent peptides; clearly evident when two (or more) samples are analyzed simultaneously. These "wide-net" projects are run on either the LTQ or Q/TOF. If the target(s) of the investigation are predetermined, however, then MRM on a 3Q can simultaneously provide high specificity and sensitivity for several compounds in the same sample. Although the technique is sometimes compared with Western Blotting, it differs in that MRM achieves a linear response (for numerous targets) over four orders of magnitude in concentration with an RSD below 10%. The LOQ can often reach low (or sub) ng/uL. These targeted, quantitative protocols require a much greater investment in development time and should only be considered after consultations with the Facility Director.
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