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Protein-resistant poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared in 150 [micro]m i.d. capillaries using novel binary porogenic solvents consisting of ethyl ether and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO copolymer with molecular weights (MWs) from 2700 to 5800. The effects of the MWs and concentrations of these surfactants in the porogenic solvent mixture on the pore properties of the resultant monoliths were investigated. Several of the monoliths showed improvements in protein and peptide separations over an extended MW range compared to monoliths synthesized using non-surfactant porogens (i.e., low MW organic liquids). The pore size distributions were examined using inverse size-exclusion chromatography (ISEC) of a select series of proteins and peptides covering a wide MW range. It was found that the best monolith had relatively large fractions of micropores (

A method for mass spectrometric peptide mapping was developed, based on hydrolysis of a solid protein by acid vapor followed by mass spectrometric analysis of the cleavage products. The method is applicable to lyophilized samples as well as proteins present in gels after separation by SDS-PAGE. The cleavage specificity was established using a number of standard proteins. Three different types of cleavages were observed: specific internal backbone cleavages at Asp, Ser, Thr, and Gly and N- and C-terminal sequence ladders. On the basis of the observed cleavage characteristics, a strategy for protein identification based on the peptide mass maps was developed. The identification strategy utilizes the specific internal backbone cleavages as well as the partial sequence information, obtained from the sequence ladders.

We report the application of aerosol matrix-assisted laser desorption ionization (MALDI) to liquid chromatography/mass spectrometry (LC/MS). The aerosol MALDI experiment uses aerosol liquid introduction in conjunction with pulsed UV laser ionization to form ions from large biomolecules in solution. Mass analysis is achieved in a time-of-flight mass spectrometer. In the LC/MALDI-MS experiment, the matrix solution is combined with the column effluent in a mixing tee. LC/MALDI-MS is demonstrated for the separation of bradykinin, gramicidin S, and myoglobin.

Selected applications of analytical techniques for the separation and analysis of peptides and proteins are reviewed. This involves the utilization of high-performance liquid chromatography, affinity chromatography, capillary electrophoresis, mass spectrometry and nuclear magnetic resonance (NMR). A separate section on the coupling of mass spectrometry to a separation method and an overview of recent developments in NMR analysis are included.

Direct control of the zeta potential and electroosmotic flow with an external electric field allows efficient separation and resolution of peptides in capillary zone electrophoresis (CZE). Electroosmotic flow controls migration rates and zone resolutions. The strength and polarity of the zeta potential shape the degree of peptide and protein adsorption via electrostatic interactions. The drop in protein and peptide adsorption at the capillary wall raises the efficiency of their separation. The effects of direct electroosmotic control are studied at various solution conditions.

This review focuses on selected applications of the separation and analysis of peptides and proteins published during the period of 1997-1998. Specific topic areas covered include high-performance liquid chromatography (HPLC), ultrafiltration, capillary electrophoresis (CE), affinity-based methods for protein isolation and separation, mass spectrometry (MS), detection of nonenzymatic posttranslational modifications, nuclear magnetic resonance spectroscopy (NMR), infrared (IR) and Raman spectroscopy, circular dichroism (CD), UV-visible absorption spectroscopy, dynamic light scattering, and calorimetry. The quantification and identification of peptides and proteins by chromatographic methods and MS have become fairly routine, as has the conformational analysis of peptides and small proteins in solution by CD, IR, and NMR. Therefore, these topics are not reviewed in detail here. In this review, we have attempted to highlight new technological developments or unique applications of analytical methods that impact the analysis of peptides and proteins.

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.4-0.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.