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

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 min separation
and improved single-shot analysis. An average of 977 protein groups
and 5605 unique peptides were identified from 50 ng of an E. coli digest, and 2158 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 h (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.