Fractionation of Complex Protein Mixture by Virtual Three-Dimensional Liquid Chromatography Based on Combined pH and Salt Steps

Abstract

The complexity and diversity of biological samples in proteomics require intensive fractionation ahead of mass spectrometry identification. This work developed a chromatographic method called virtual three-dimensional chromatography to fractionate complex protein mixtures. By alternate elution with different pHs and salt concentrations, we implemented pH and salt steps by turns on a single strong cation exchange column to fully exploit its chromatographic ability. Given standard proteins that were not resolved solely by pH or salt gradient elution could be successfully separated using this combined mode. With a reversed phase column tandem connected behind, we further fractionated as well as desalted proteins as the third dimension. This present strategy could readily be adapted with respect to special complexity of biological samples. Crude plasma without depleting high abundance proteins were fractionated by this three-dimensional mode and then analyzed by reversed phase liquid chromatography coupled with LTQ mass spectrometry. In total, 1933 protein groups with wide dynamic ranges were identified from a single experiment. Some characteristics that correlated to the behavior of proteins on strong cation exchange columns are also discussed.

Analysis of Proteome Profile in Germinating Soybean Seed, and Its Comparison with Rice Showing the Styles of Reserves Mobilization in Different Crops

“…The output results were combined by BuildSummary software with a false positive rate (FPR) less than 1% as the filtration criterion. The FPR was calculated as double of the number of peptides from reversed database divided by the number of peptides from reversed and normal database [46]. The protein identification criteria were based on Delta CN (≥0.1) and cross-correlation scores (Xcorr, one charge≥1.9, two charges ≥2.2, three charges ≥3.75).…”

Mass spectrometry of peptides and proteins from human blood

“…Blood samples have poor rheological properties that may lead to high back pressures, column fouling, and drastically shortened analytical column life . In contrast to analytical chromatography that typically relies on a binary buffer system, batch chromatography samples are bound to the stationary phase of the column and eluted in one or many different fractions by a step-gradient of ionic strength or pH Guerrier, Lomas, & Boschetti, 2007;Guerrier et al, 2008;Kreusch et al, 2008;Ning et al, 2008;Tucholska et al, 2009). Separation of intact proteins using a novel library of peptides (Guerrier et al, 2006) as a stationary phase was observed to work well (Baussant et al, 2005;Sennels et al, 2007;Sihlbom et al, 2008).…”

“…Partition chromatography has a high sample capacity and the resolving power of the column depends on its length, the number of plates, the relative amount of sample loaded, plus the selectivity of the different loading and eluting buffers used in the experiment (Ning et al, 2008;Tucholska et al, 2009). Many proteins were purified from blood fluids using sequential chromatography (Duckert, Koller, & Matter, 1953;Kanai, Raz, & Goodman, 1968;McConnell & Mason, 1970;Murthy & Hercz, 1973;Poffenbarger, 1975;Tollefsen, Majerus, & Blank, 1982).…”

Peptide fractionation in proteomics approaches

“…Further extensions of multiple chromatographic approaches (3D-LC [36]) can increase LC-MS time to the point where stability of the system might become an issue;…”