Potential of long capillary monolithic columns for the analysis of protein digests

Abstract

The gain in separation efficiency for protein digests using long monolithic columns has been evaluated for a LC-MS system with capillary monolithic columns of different lengths (150 and 750 mm). A mixture of BSA, alpha-casein and beta-casein tryptic digests was used as a test sample. Peak capacity and productivity (peak capacity per unit time) were determined from base peak chromatograms and MS/MS data were used for protein identification by MASCOT database searching. Peak capacity and protein identification scores were higher for the long column. Analyses with similar gradient slope for the two columns produced ratios of the peak capacities that were slightly higher than the expected value of the square root of the column length ratio. Peak capacity ratios varied from 2.7 to 4.0 for four different gradient slopes, while protein identification scores were 2-4 times higher for the long column. Similar values were obtained for the productivity of both columns and the highest productivity was obtained at gradient times of 45 and 75 min for the short and long column, respectively. The use of long monolithic columns improves peptide separation and increases reliability of protein identification for complex digests, especially if longer gradients are chosen.

Potential of poly(styrene-co-divinylbenzene) monolithic columns for the LC-MS analysis of protein digests

“…However, there does not seem to be any indication that C18-silica monoliths outperform the PS-DVB columns, as suggested in literature [27]. While the peak capacities obtained for the C18-silica monolith in [27] are similar to those we found earlier [16], the authors find much lower values for the PS-DVB column. A peak capacity of 65 was reported for a gradient of 0.25% ACN min −1 , whereas the peak capacity at such a gradient would be around 170 in our system.…”

“…Comparison of peak capacities for the short (30 min gradient) and long (150 min gradient) columns results in a peak capacity ratio of 3.36, which is about 50% higher than the expected value of . This number is comparable to results obtained in the comparison of C18-silica monoliths of different length [16]. …”

“…The efficiency can be optimized further by tuning the morphology, while covalent attachment of the monolith against the fused-silica capillary wall ensures high robustness and eliminates channelling effects. While the advantages of using longer columns have been proven for silica-based monoliths [12–16], the use of polymer-based monoliths has been limited to the application of short (5–6 cm) PS-DVB-based columns for fast separations of relatively simple mixtures [7, 17, 18]. …”

Performance evaluation of long monolithic silica capillary columns in gradient liquid chromatography using peptide mixtures

“…In the present contribution reversed- phase peptide separations obtained through UV-detection will be illustrated while applying a commercial nano-LC system at conventional pressures (below 400 bar). In almost all previous studies dealing with gradient elution, silica monoliths were exclusively evaluated or used in combination with MS [11][12][13]. This approach is of course much more favorable when aiming towards the identification of unknown components.…”

Recent advances in monolithic columns for protein and peptide separation by capillary liquid chromatography

“…Various kinds of monolithic capillary columns have been synthesized for RPLC separation of proteins and peptides, such as poly(styrene-co-divinylbenzene) monoliths [53][54][55][56], poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths [57], poly(lauryl methacrylateco-ethylene dimethacrylate) monoliths [58,59], monoliths derived from ring-opening metathesis polymerization [60], C 18 -modified silica monoliths [61][62][63][64][65][66], dimethyldodecylammonium-silica hybrid monoliths [44], POSS-based hybrid monoliths [49], and phenyl-silica hybrid monoliths [47]. The detailed applications are shown in Table 1.…”