MD-LC/MS methods

One may argue that the number of 1000 proteins in a biofluid sample is still far too small. Comprehensive methods, such as 2D PAGE or MD-LC MS are generally known to be rather time-consuming, making them more suitable for initial discovery efforts than for larger clinical validation studies. In the end, it may not be necessary to visualize every low-abundance protein in order to find significant differences that lead to novel markers. 

Pure reference standards of B complex vitamins can be obtained from USP. Once a LC-MS method for pantothenic acid analysis from multivitamin dietary supplements is developed, the multi-element multivitamin dietary supplement Standard Reference Material 3280 (SRM 3280) from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) can be used for validation and quality control. 

A number of proteomic studies on archival material have utilized Liquid Tissue (Expression Pathology, Inc., Gaithersburg, MD), a commercial protein extraction kit for FFPE tissue.4,9,25-28 This kit is also based upon HIAR techniques and shares a similar work flow to the methods already discussed. Thin, typically 5-10pM, sections are cut from paraffin tissue blocks, the paraffin is removed, and the tissue deparaffinized and rehydrated in alcohols and distilled water before microdissection. The cellular material is then suspended in Liquid Tissue buffer and heated at 95°C for 90 min. Trypsin is added, and the material is digested overnight at 37°C prior to reduction with DTT and analysis by LC-MS/MS.26... 

Method development for mass spectrometry portion of LC-MS/MS assay is relatively simple and straightforward. For an experienced method development (MD) scientist, the optimization typically takes only hours to complete. In contrast, method development for optimal liquid chromatography conditions can be one of the most challenging tasks. Chromatography development can be very time-consuming. The task is further complicated by the nearly infinite choices in chromatography options such as vendor, sorbent, solvent selection, particle size, and column dimensions. 

Therefore, the H/D exchange method was evaluated by the authors to differentiate between these possibilities. The online H/D exchange LC—MS experiment using D2O in the mobile phase afforded [Md + D ] ions at m/z 302 and 303 for Ml (Fig. 10.9c) and M2 (Fig. 10.10c), respectively. This revealed that Ml had no exchangeable hydrogen atom in its structure, which ruled out... 

The LC-MS/MS instrumental detection limits were roughly estimated hy extrapolation of S/B values measured for the clean standard solutions to S/B = 3, and comparisons of the results obtained using different instruments nicely illustrates the advances in ESI—MS/MS technology over the last 20 years or so. The LOD values for the 20 toxins obtained using optimized MRM on the first commercial LC-MS/MS instrument (MDS-SCIEX API in, manufactured 1988) were in the range 40-7000 nM for an injection volume of 5 p,L, but when a modem version (API 4000) was used under identical conditions the LOD range was 5-30 nM, that compares favorably with values 5-100 uM obtained (Oshima 1995) for the LC-oxidation-fluorescence method. 

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