Surface-Confined Aqueous Reversible Addition–Fragmentation Chain Transfer (SCARAFT) Polymerization Method for Preparation of Coated Capillary Leads to over 10 000 Peptides Identified from 25 ng HeLa Digest by Using Capillary Zone Electrophoresis-Tandem Mass Spectrometry

Abstract

A surface-confined aqueous reversible addition-fragmentation chain transfer (SCARAFT) polymerization method was developed to coat capillaries for use in capillary zone electrophoresis (CZE). SCARAFT polymerization primarily takes place on the inner surface of the capillary instead of in solution, which greatly improves the homogeneity of the coating. Capillaries treated with this coating produced an electroosmotic mobility of 2.8 ± 0.2 × 10−6 cm2·V−1·s−1 (N=3), which is roughly an order of magnitude lower than that of commercial linear polyacrylamide (LPA) coated capillaries. Coated capillaries were evaluated for bottom-up proteomic analysis using CZE. The very low electroosmotic mobility results in a 200-minute separation and improved single-shot analysis. An average of 977 protein groups and 5,605 unique peptides were identified from 50 ng of an E. coli digest and 2,158 protein groups and 10,005 peptides were identified from 25 ng of a HeLa digest using single-shot analysis with a SCARAFT-acrylamide capillary coupled to a Q Exactive HF mass spectrometer. The coating is stable. A single capillary was used for over 200 hours (8.4 days) of continuous operation. RSD in migration time was between 2 and 3% for selected ion electropherograms (SIEs) generated for six ions; median theoretical plate counts ranged from 240,000 to 600,000 for these SIEs. Various types of coatings could be prepared by simply changing the functional vinyl monomers in the polymerization mixture. Positively charged coatings using direct attachment and formation of a block-copolymer were prepared and demonstrated for the separation of mixtures of intact proteins.

Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms

“…We measured the electroosmotic mobility in the LPA-coated capillary based on the method reported in literature. [42,43] The electroosmotic mobility in 10% (v/v) acetic acid BGE was lower than that in 0.1% (v/v) formic acid BGE (6.8×10 −6 cm 2 ·V −1 ·s −1 vs. 1.1×10 −5 cm 2 ·V −1 ·s −1 ). The details about measurement of the electroosmotic mobility is described in Supporting Information I.…”

Production of Over 27 000 Peptide and Nearly 4400 Protein Identifications by Single-Shot Capillary-Zone Electrophoresis–Mass Spectrometry via Combination of a Very-Low-Electroosmosis Coated Capillary, a Third-Generation Electrokinetically-Pumped Sheath-Flow Nanospray Interface, an Orbitrap Fusion Lumos Tribrid Mass Spectrometer, and an Advanced-Peak-Determination Algorithm

“…A published protocol was use for coating the capillary. 8 Briefly, a piece of ∼110 cm long, 50-μm ID, 360 μm OD fused silica capillary was pretreated by flushing with 0.1 M NaOH for 2 h, flushing with water until the outflow reached pH ∼7.0, flushing with 0.1 M HCl for 12 h, flushing again with water until the outflow reached pH ∼7.0, and drying with a nitrogen stream overnight at room temperature. Then, the capillary was filled with 50% cyanomethyl [3-(trimethoxysilyl)propyl] trithiocarbonate solution in MeOH(v/v) and incubated in a water bath at 45 °C for 12 h. The capillary was then rinsed with MeOH to flush out the residual reagent and dried with a nitrogen stream.…”

“…9–15 To date, the best single shot CZE-ESI-MS/MS bottom-up proteomic analyses of eukaryotic systems have identified approximately 10,000 peptides and 2,000 proteins. 8, 10…”

Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (2015–mid 2018)

“…Recently, a novel surface‐confined aqueous reversible addition‐fragmentation chain transfer (SCARAFT) polymerization method for capillary coating, where polymerization occurs mainly on the inner surface of the capillary enhancing thereby the homogeneity of the coating, was evaluated by CZE‐MS analysis of tryptic digests and protein mixtures .…”

“…Recently, a novel surface-confined aqueous reversible addition-fragmentation chain transfer (SCARAFT) polymerization method for capillary coating, where polymerization occurs mainly on the inner surface of the capillary enhancing thereby the homogeneity of the coating, was evaluated by CZE-MS analysis of tryptic digests and protein mixtures [115]. The sequence specific model was developed to predict peptide electrophoretic mobility for an extended set of tryptic peptides (4000) identified by CZE-MS/MS [116].…”