Post-polymerization photografting on methacrylate-based monoliths for separation of intact proteins and protein digests with comprehensive two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry

Post-polymerization photografting is a versatile tool to alter the surface chemistry of organic-based monoliths so as to obtain desired stationary phase properties. In this study, 2-acrylamido-2-methyl-1-propanesulfonic acid was grafted to a hydrophobic poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith to create a strong cation exchange stationary phase. Both single-step and two-step photografting were addressed, and the effects of grafting conditions were assessed. An experimental design has been applied in an attempt to optimize three of the key parameters of the two-step photografting chemistry, i.e. the grafting time of the initiator, the monomer concentration and the monomer irradiation time. The photografted columns were implemented in a comprehensive two-dimensional column liquid chromatography (tLC × tLC) workflow and applied for the separation of intact proteins and peptides. A baseline separation of 11 intact proteins was obtained within 20 min by implementing a gradient across a limited RP composition window in the second dimension. tLC × tLC with UV detection was used for the separation of cytochrome c digest, bovine serum insulin digest and a digest of a complex protein mixture. A semi-quantitative estimation of the occupation of separation space, the orthogonality, of the tLC × tLC system yielded 75 %. The tLC × tLC setup was hyphenated to a high-resolution Fourier transform ion cyclotron resonance mass spectrometer instrument to identify the bovine serum insulin tryptic peptides and to demonstrate the compatibility with MS analysis. Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8615-4) contains supplementary material, which is available to authorized users.


Aim
In this article we tried to optimize the two-step post-polymerization photografting conditions of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) to a monolithic butyl methacrylate copolymerized with ethylene glycol dimethacrylate (poly(BMA-co-EDMA)). Two different polymerization batches were investigated, details can be found in Table S1. Parameters to optimize the photografting conditions were selected based via an experimental design. Three grafting parameters, of the two-step post-polymerization photografting, were chosen to yield in the highest bindingcapacity of the resin (i.e. the grafting time of the initiator (t 1 ), the monomer concentration (c m ) and the monomer irradiation time (t 2 )). All 15 different grafting conditions can be found in Table S2. The photgrafted columns were implemented in comprehensive twodimensional liquid chromatography for the analyzes of proteins and protein digest. Finally we demonstrated the ability to hypenate the setup with a high-resolution Fourier-transform ioncyclotron mass spectrometor (FTICR-MS/MS) for the analyses of a BSA digest.

Modeling of the photografting conditions using an experimental design
The quantitative breakthrough of BTMAC (50% height) was used as the response for the modelling. In Fig. S1A a sub-set of three grafting conditions (1-3, identical to conditions from Fig. 3B) were compared to the breakthrough capacity of the maximum predicted optimum condition (t 1 = 3.4 min, t 2 = 6.4 min, c m = 8.1%) (5). This resulted in an experimental capacity which was higher than the predicted experimental capacity (4) (Y exp = 100 µg per mL column volume vs. Y pred = 65 ± 40 µg per mL column volume). We applied the quadratic model, without interaction, to calculate the predicted binding capacities at the various grafting conditions (Fig.   S1C). The yellow color indicates high Y pred and the grafting conditions resulting in the blue region result in a low Y pred . * percentages of initiator with respect to the monomer concentration.

Fig. S1
Determination of grafting yield by the breakthrough of curves at varied monomer grafting time Calculated grafting yield at all grafting conditions 1) t 1 = 4 min, t 2 = 2 min, c m = 7.5%, 2) min, c m = 7.5%, 4) theoretical breakthrough of optimal grafting point and 5) experimental breakthrough of optimal grafting point S4 Determination of grafting yield by the breakthrough of BTMAC onomer grafting times (t 2 ). B) predicted vs. experimental binding capacity. eld at all grafting conditions within the experimental design = 7.5%, 2) t 1 = 4 min, t 2 = 7 min, c m = 7.5% and 3 ) 4) theoretical breakthrough of optimal grafting point and 5) experimental rough of optimal grafting point BTMAC. In A) Breakthrough experimental binding capacity. C) within the experimental design. = 7.5% and 3 ) t 1 = 4 min, t 2 = 12 4) theoretical breakthrough of optimal grafting point and 5) experimental

Hyphenation of LC×LC with
In Fig. S2 the valve configuration is shown that was used for comprehensive two combined with the FTICR-MS.
the left valve accordingly. The collected fractions were injected in the desalting was applied by trapping the peptides salt residue to waste. The 2 D effluent was split flow did not exceed 500 nL·min   The chromatograms were with 5 min isocratic elution at 0.5 mM KCl followed by a increase to 249.5 mM in 60 min at a in 9.0 min, with 3 min de-salting and