>>J Chromatogr A. 2005 May 6;1073(1-2):25-33.
A simplified monobuffer multidimensional chromatography for high-throughput proteome fractionation.
Guerrier L, Lomas L, Boschetti E.
Department of Biology Research, Ciphergen, 6611 Dumbarton Circle, Fremont, CA 94555, USA. lguerrier@ciphergen.com
The complexity of the human serum proteome is attributed to both a large dynamic range of protein abundance, as much as 10 orders of magnitude, and a disproportionate few dozens of proteins representing as much as 99% of the total protein content. These characteristics make it beneficial to use a pre-fractionation step prior to any high-resolution analysis, such as mass spectrometry. The present method describes a unimodal multidimensional chromatography concept to rapidly achieve an effective fractionation of human serum that is directly amenable with surface-enhanced laser desorption/ionization (SELDI)-based mass spectrometry. This method is based on the use of a column composed of a superimposed sequence of sorbents. The assembly is first equilibrated with a single binding buffer and then loaded with the whole crude sample. As the sample crosses the different adsorbent layers proteins within are sequentially trapped according to the complementary properties vis-a-vis of the sorbent. Once the loading and capturing is achieved, the sequence of columns is disassembled and each column, containing different complement of proteins is eluted separately in a single step and under optimal elution conditions. When compared to classical single-chemistry fractionation based on, for example, anion-exchange and pH stepwise elution, the new proposed approach shows much lower protein overlap between fractions, and therefore, greater resolution. This results in a larger number of detectable species, and therefore, reinforces the power of discovery of new biomarkers. A significantly higher sensitivity for low-abundance species was additionally found as evidenced by spiking trials.<<
Further work on simplifying the complexity of the "matrix" that is human serum, which some MS users apparently feel limits diagnostic utility because of noise from all the extra stuff.
Cheers, Tuck |
