Protein/peptide analysis reversed-phase chromatography

YF Maa, Cs Horvath. Rapid analysis of proteins and peptides by reversed-phase chromatography with polymeric micro-pellicular sorbents. J Chromatogr 445 71-86, 1988. 

The effect of mobile phases on the sensitivity in the analysis of peptides and proteins by HPLC coupled with electrospray mass spectrometry has been reviewed (63). The analysis of proteins and peptides using reversed phase chromatography mostly involves the use of trifluoroacetic acid as an ion-pairing agent despite this compound being a strong suppressor of the mass spectroscopic signal. 

Figure 2. Workflow of an LC-MS/MS experiment. A mixture of peptides from a protein sample digest is separated by reversed-phase chromatography on a nano-flow HPLC. The peptides elute from the RP column and are ionized by an electrospray source. In the first stage of mass spectrometry, m/z values and charge states for each precursor ion are determined and the most abundant precursor ions are selected for analysis in the second stage. The ions are then fragmented with by collision-induced dissociation (CID) a gas to produce fragment ions which are detected. Using the mass (from MS-1) and sequence information (from MS-2) protein sequence databases are searched to provide peptide identifications and protein matches.

The rapid advancement in peptide research over the past 25 years must be attributed, in part, to the effectiveness of high-performance liquid chromatography (HPLC), particularly reversed-phase chromatography, in the separation and analysis of peptides. The resolution and selectivity of this technique allows peptides to be effectively isolated and purified from closely related substances. It also separates most or all of the components of complex biological mixtures such as tryptic digests of proteins. 

Reversed phase chromatography is often used for the analysis of peptides and other relatively small biomolecules. Other kinds of liyciropliohie interaction columns, using polysaccharide supports modified with less hydrophobic groups (phenyl groups, or shorter hydrocarbons), are used for protein separations. 

Various applications of PITC in analyses of acid hydrolysates of purified proteins and peptides (91) and of hydrolysates of feeds (92) have been described. Bidlingmeyer et al. (93) showed reversed-phase chromatographic analysis of free amino acids and hydrolysates of foods (soybean flour, mozzarella cheese, beer, and soy sauce) with formation of PITC derivatives to be a fast, reproducible method that presented a very good correlation with the results obtained by ion-exchange chromatography. 

Reversed-phase HPLC is widely utilized to generate a peptide map from digested protein, and the MS online method provides rapid identification of the molecular mass of peptides. The HPLC-MS-FAB online system is a sensitive and precise method for low-MW peptides (<3000 Da) even picomol quantities can be detected. However, as the MW of the analytes increases, the ionization of peptides becomes more difficult and decreases the sensibility of the FAB-MS (112). Electrospray ionization (ESI-MS) was found to be an efficient method for the determination of molecular masses up to 200,000 Da of labile biomolecules, with a precision of better than 0.1%. Molecular weights of peptide standards and an extensive hydrolysate of whey protein were determined by the HPLC-MS-FAB online system and supported by MALDI-TOF (112). Furthermore, HPLC-MS-FAB results were compared with those of Fast Performance Liquid Chro-motography (FPLC) analysis. Mass spectrometry coupled with multidimensional automated chromatography for peptide mapping has also been developed (9f,l 12a). 

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