Monolith tryptic digestion

FIGURE 7.13 Two-dimensional separation of tryptic digest of BSA in simple 2D-HPLC. Capillary monolithic silica-C18 column (0.1 mmi.d., 10 cm) was used as 2nd-D column. Mobile phase for 2nd-D gradient started with 0% B at 0.5 min, increased to 50% B at 3.3 min, to 100% B at 3.5 min, then returned to the initial condition and held for the last 0.5 min. Flow rate 3.0 pL/min in capillary, and 2 mL/min at the pump. Other conditions are similar to those for Figure 7.11 (reproduced from the reference, Kimura et al. (2004) with permission from Wiley). 

Ivanov, A.R., Zang, L., Karger, B. L. (2003). Low-attomole electrospray ionization MS and MS/ MS analysis of protein tryptic digests using 20 pm-i.d. polystyrene-divinylbenzene monolithic capillary columns. Anal. Chem. 75, 5306-5316. 

The main benefit of monolithic columns is the separation speed. As an example of the practical use of ultrafast separation, the two-dimensional HPLC separation of tryptic digest BSA is shown. The fractions of the column effluent from the first dimension (lEX) separation was collected for 2 min each and injected into the second-dimension monolithic column (RP).The run time for the second dimension could not be more than 2 min (more than fraction collection time), and the monolithic column provided fast and efficient separation (Figure 3-26). 

Figure 3-26. Two-dimensional separation of tryptic digest of BSA by simple 2D-HPLC. First-D cation exchange separation (horizontal axis), MCI CQK-31S 5-mm (50 mm, 2.1-mm i.d.) column, 2nd-D reversed-phase separation (vertical axis), monolithic silica-C18 Chromolith Flash (25 mm, 4.6 mm i.d.) column. The fractionation at the Ist-D was done every 2min. (Reprinted from reference 98, with permission.)...

Prefractionation of proteins in an Arabidopsis thalicma leaf extract by means of fast-performance LC prior to SCXxRPLC-MS on a monolithic RPLC column allowed the identification of 1032 unique proteins in 4 mg of a protein plant leaf tissue extract [59]. Fractionation of human plasma proteins by on-line SCX and RPLC was performed prior to tryptic digestion [60]. The resulting 30 fractions were digested and analysed by LC-MS on a quadrupole-linear-ion-trap (Q-LIT) instmment. Using this approach, 1292 proteins were identified, some of which are know to be present at <10 ng/ml. 

FIGURE 47.6 Schematic diagram of the microfluidic device fabricated from cyclic olefin copolymer containing poly(ethylhexyl methacrylate-co-ethylene dimethacrylate) monolith (top) and chromatogram of tryptic digested bovine serum albumin in the reversed-phase mode. Conditions digest sample 5 pmol/ xL, mobile phase A 5% acetonitrile in 0.1% aqueous TFA, mobile phase B 70% acetonitrile in 0.1% aqueous TEA, gradient 0% B for 5 min, then from 0% to 70% B in 60 min, flow rate 300 nL/min. 

FIGURE 47.14 Time-of-flight mass spectra of peptides prepared via tryptic digestion of bovine hemoglobin. (Reprinted with permission from Lazar, I. M., et al., Electrophoresis, 24, 3655, 2003. Copyright 2003 Elsevier B.V.) (a) Peptides separated in the CEC system with monolithic column shown in Figure 47.13, (b) unseparated... 

Similarly, Huang et al. reported the separation of standard proteins, tryptic digests of cytochrome c, and myoglobin on both macroporous underivatized and octadecylated PS/DVB monoliths. 

Figure 4 Separation and mass analysis of tryptic peptides of bovine catalase. Column, monolithic PS/DVB, 60 x 0.20mm i.d. mobile phase (A) 0.050% TFA in water (B) 80% acetonitrile, 0.050% TFA in water linear gradient, 2-60% B in 15 min flow rate, 1.8 pi min temperature, 50 C scan, m/z 400-2000 electrospray voltage, 4.0 kV sample, 5 pmol of tryptic digest of bovine catalase. (Reprinted with permission from Premstaller et al. (2001) Analytical Chemistry 73 2392-2994 2003 American Chemical Society.)...

Literature on enzyme microreactors for chemical synthesis is scarce. There is abundant literature, however, on the use of enzymes in microsystems for purposes of DNA analysis, e.g. using the polymerase chain reaction discussed before or DNA restriction fragment analysis [81], for proteomics, such as tryptic digestion of proteins with the enzyme free in solution [82], with trypsin-coated beads trapped in a microreactor [83] or trypsin immobilized on a porous polymer monolith (in a fused-silica capillary) [84]. Also, enzyme microreaction devices have been used extensively for medical diagnostics or immunoassays, e.g. using porous silicon as a support for immobilized enzymes [85]. 

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