Multiplexed Two-Dimensional Liquid Chromatography for MALDI and Nanoelectrospray Ionization Mass Spectrometry in Proteomics

Abstract

We developed a multiplexed two-dimensional separation system based on reversed phase (RP)--strong cation exchange (SCX) chromatography as a front-end device for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization (nanoESI) mass spectrometry. Tryptic peptide mixtures were fractionated on a reversed-phase HPLC column, and each fraction was loaded onto multiplexed SCX microcolumns. Because this second chromatography was carried out in parallel, the analysis time is independent of the fraction number in the first RP-HPLC separation. The resultant samples were desalted/concentrated and eluted onto a MALDI plate with matrix-containing elution solutions in parallel, or eluted with optimized solutions for nanoESI and loaded onto nanoESI sprayers by an automated instrument. The soluble portion of HCT116 lysate was digested and fractionated using a 48-plexed chromatography system. Approximately 1000 unique peaks were detected in MALDI-MS with 3000 MS/MS spectra, while 724 peptides with ultrahigh peptide mass accuracy (sub-ppm error) were identified in nanoESI-FTICR mass spectrometry with five integrated selected ion monitoring scans. Since MS measurement with this off-line LC-LC approach is not restricted by continuous LC elution, it is expected to be useful especially in cases where repeated analysis with different scan modes or long-term data acquisition is required.

Evaluation of the multivariate selectivity of multi‐way liquid chromatography methods

“…This number of components is also consistent with that seen in proteomics data sets, which can contain from hundreds to thousands of components [8,10,33,34]. The retention times of these 500 peaks were randomly varied within the defined separation space over 50 iterations.…”

Phosphorylation of BLUS1 kinase by phototropins is a primary step in stomatal opening

“…TCA-treated guard cell proteins (100 mg) were suspended in 0.1 M Tris-HCl (pH 8.0) containing 8 M urea, protein phosphatase inhibitors and a protease inhibitor cocktail (Sigma-aldrich), and the suspension was sonicated for 5 min. The proteins were reduced by dithiothreitol, alkylated with iodoacetamide and digested with Lys-C, followed by trypsin treatment 37 . The digested samples were desalted using stage tips with C18 Empore disk membranes (3M) 38 .…”

Large-Scale Comparative Phosphoproteomics Identifies Conserved Phosphorylation Sites in Plants

“…The homogenate was centrifuged at 1,500g for 10 min, and the supernatant was added with urea at a final concentration of 8 M. The protein amount in the solution was measured with a bicinchoninic acid protein assay kit (Thermo Scientific). The solution was reduced with 10 mM dithiothreitol for 30 min at room temper-ature, alkylated with 50 mM iodoacetamide for 30 min at room temperature in the dark, and digested with Lys-C (1:100, w/w) for 3 h at room temperature, followed by dilution 4-fold with 50 mM ammonium bicarbonate and digestion with trypsin (1:100, w/w) overnight at room temperature (Saito et al, 2006). These digested samples were acidified with the addition of trifluoroacetic acid (TFA) and were desalted using StageTips with C18 Empore disc membranes (3M; Rappsilber et al, 2003) as described below.…”