Capillary Isoelectric Focusing-Tandem Mass Spectrometry and Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry for Quantitative Proteomic Analysis of Differentiating PC12 Cells By Eight-Plex Isobaric Tags for Relative and Absolute Quantification

Abstract

We report the application of capillary isoelectric focusing for quantitative analysis of a complex proteome. Biological duplicates were generated from PC12 cells at days 0, 3, 7, and 12 following treatment with nerve growth factor. These biological duplicates were digested with trypsin, labeled using eight-plex iTRAQ chemistry, and pooled. The pooled peptides were separated into 25 fractions using reversed-phase liquid chromatography (RPLC). Technical duplicates of each fraction were separated by capillary isoelectric focusing (cIEF) using a set of amino acids as ampholytes. The cIEF column was interfaced to an Orbitrap Velos mass spectrometer with an electrokinetically-pumped sheath-flow nanospray interface. This HPLC-cIEF-ESIMS/MS approach identified 835 protein groups and produced 2,329 unique peptides IDs. The biological duplicates were analyzed in parallel using conventional strong-cation exchange (SCX) – RPLC-ESIMS/MS. The iTRAQ peptides were first separated into eight fractions using SCX. Each fraction was then analyzed by RPLC-ESI-MS/MS. The SCX-RPLC approach generated 1,369 protein groups and 3,494 unique peptide IDs. For protein quantitation, 96 and 198 differentially expressed proteins were obtained with RPLC-cIEF and SCX-RPLC, respectively. The combined set identified 231 proteins. Protein expression changes measured by RPLC-cEIF and SCX-RPLC were highly correlated.

Mixed-mode chromatographic stationary phases: Recent advancements and its applications for high-performance liquid chromatography

“…With n-octyltrimethoxysilane (C 8 -TMOS) and vinylphosphonic acid as functional monomers, a one-step polycondensation and simultaneous "thiol-ene" click chemistry were further used to prepare C 8 /PO(OH) 2 Furthermore, monolithic-based mixed-mode stationary phases have been less reported. Thus, we anticipate that the development of novel mixed-mode monolithic columns will be another hot-spot in the future research.…”

“…Up till now, HPLC has become one of the most popular and powerful techniques in separation science, widely used in separation, qualitative and quantitative determination in a variety of fields [1][2][3][4][5][6].…”

“…Schematic diagram for preparation of V-shape bis(tetraoxacalix[2]arene[2]triazine) modified silica-based stationary phase. (Reprinted from[58]).…”

“…Furthermore, the developed stationary phase can be used for rapid separation and detection of illogical additive of melamine in powdered infant formula in the mobile phase without addition of ion-pair reagents or buffer solution. In the same group, a V-shape bis(tetraoxacalix[2]arene[2]triazine) modified silica-based stationary phase was further prepared, as shown in…”

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for quantitative parallel reaction monitoring of peptide abundance and single-shot proteomic analysis of a human cell line

“…The interface has several advantages, including minimal sample dilution due to the very low sheath flow rate, elimination of mechanical pumps and nebulizing gas, use of a wide range of separation buffers, and stable operation in the nanospray regime. We have applied the electrokinetically pumped sheath flow nanospray interface CE-MS/MS system for shotgun proteomic analysis of the secretome of Mycobacterium marinum [18], a fraction of yeast lysate [19], the E.coli proteome [20-22], picogram amounts of RAW 264.7 cell lysate [23-25], and the PC12 cell lysate [26]. In addition, the system was also applied for top-down intact protein characterization [27], quantitative multiple reaction monitoring (MRM) of peptide abundance [28], and phosphopeptides characterization [29].…”