Poly(acrylamide- co- N, N'-methylenebisacrylamide) Monoliths for High-Peak-Capacity Hydrophilic-Interaction Chromatography-High-Resolution Mass Spectrometry of Intact Proteins at Low Trifluoroacetic Acid Content

Abstract

In this study, we optimized a polymerization mixture to synthesize poly(acrylamide-co-N,N'-methylenebisacrylamide) monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of cross-linker and the porogen composition on the separation performance of the resulting columns were studied. The homogeneity of the structure and the different porosities were examined through scanning electron microscopy (SEM). Further characterization of the monolithic structure revealed a permeable (K_f between 2.5 × 10^-15 and 1.40 × 10^-13 m²) and polar stationary phase suitable for HILIC. The HILIC separation performance of the different columns was assessed using gradient separation of a sample containing four intact proteins, with the best performing stationary phase exhibiting a peak capacity of 51 in a gradient of 25 min. Polyacrylamide-based materials were compared with a silica-based particulate amide phase (2.7 μm core-shell particles). The monolith has no residual silanol sites and, therefore, fewer sites for ion-exchange interactions with proteins. Thus, it required lower concentrations of ion-pair reagent in HILIC of intact proteins. When using 0.1% of trifluoroacetic acid (TFA), the peak capacities of the two columns were similar (30 and 34 for the monolithic and packed column, respectively). However, when decreasing the concentration of TFA to 0.005%, the monolithic column maintained similar separation performance and selectivity (peak capacity 23), whereas the packed column showed greatly reduced performance (peak capacity 12), lower selectivity, and inability to elute all four reference proteins. Finally, using a mobile phase containing 0.1% formic acid and 0.005% TFA, the HILIC separation on the monolithic column was successfully hyphenated with high-resolution mass spectrometry. Detection sensitivity for protein and glycoproteins was increased and the amount of adducts formed was decreased in comparison with separations performed at 0.1% TFA.

Figure 1

SEM images of cross sections of three different acrylamide-based monolithic capillary columns. (A) 25DMF, (B) A50, and (C) A55. Scale bars, 10 μm.

Figure 2

HILIC separation of intact proteins (A–C) on three capillary columns (A50, A55, and 25DMF; top to bottom of 0.2 mm ID × ca. 150 mm). The analysis was performed using a gradient from 90 to 85% (v/v) B in 1 min and then down to 55% B in 25 min at a flow rate of 2 μL/min. The analysis was carried at 60 °C, and the UV wavelength monitored was 214 nm. The chromatograms obtained with all the columns in Table 1 are shown in Figure S4 of the SI.

Figure 3

Comparison of HILIC separations of four intact proteins on the particle-packed column (0.2 mm ID × 135 mm L; black) and on the monolithic column (0.2 mm ID × 140 mm L; red). The analyses were performed using a gradient from 94 to 83% (v/v) B in 1 min and then down to 65% B in 26 min with 0.1% (v/v) TFA (A) and with 0.005% (v/v) TFA (B) in the mobile phases. Protein elution order: CC, CA, BSA, and Tf. All the analyses were carried out at 45 °C using a flow rate of 2 μL/min, and the monitored UV wavelength was 214 nm.

Figure 4

TIC (a, c) and EIC (b, d) chromatograms obtained using a HILIC–MS method with (a, b) 0.1% (v/v) TFA and (c, d) 0.005% (v/v) TFA + 0.1% (v/v) FA. (a) Gradient from 94 to 83% (v/v) B in 2 min and then down to 67% in 35 min. (b–d) Gradient from 94 to 87% (v/v) B in 2 min and then down to 70% in 35 min. All the analyses were carried at 45 °C using a flow rate of 1.5 μL/min. The EIC chromatograms were obtained by summing the intensities of different charge states for CC (883.8 , 951.7, 1030.9, 1124.4, 1236.8, 1374.1, 1545.7, 1766.2, and 2060.5 m/z), CA (937.3, 968.4, 1001.9, 1037.6, 1076.6, 1117.3, 1161.9, 1210.4, 1262.8, 1320.3, 1383.0, 1452.3, 1528.5, 1613.4, and 1708.2 m/z), BSA (1278.5, 1303.5, 1329.6, 1356.6, 1384.9, 1414.4, 1445.1, 1477.2, 1510.7, 1545.8, 1582.0, 1621.2, 1664.5, and 1704.2 m/z), and Tf (2040.8, 2094.5, 2151.1, 2210.8, 2273.9, 2340.8, 2411.7, 2487.0, 2567.2, 2652.7, and 2744.2 m/z). All the EICs are ±0.2 m/z

Figure 5

Average spectrum from the chromatographic peak of (a) CC, (b) CA, (c) BSA, and (d) Tf obtained using the HILIC–MS method presented in Figure 4. The 0.1% (v/v) TFA (black trace) and 0.005% (v/v) TFA + 0.1% (v/v) FA (red trace). The numbers on the mass spectra indicate the charge-state interval.