A simplified monobuffer multidimensional chromatography for high-throughput proteome fractionation.
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.
Guerrier
L
L
Department of Biology Research, Ciphergen, 6611 Dumbarton Circle, Fremont, CA 94555, USA. lguerrier@ciphergen.com
Lomas
L
L
Boschetti
E
E
D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry; D002021:Buffers; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D001798:Blood Proteins; D002021:Buffers; D020543:Proteome