Protein/peptide analysis HPLC methods

The specification development process is a data-driven activity that requires a validated analytical method. The levels of data needed include assay precision, replicate process results (process precision), and real-time stability profiles. A statistical analysis of these data is critical in setting a realistic specification. Most often, aggregation and fragmentation degradation mechanisms are common to protein and peptide therapeutics. Therefore, the SE-HPLC method provides a critical quality parameter that would need to be controlled by a specification limit. 

Various important LC methods for amino acid, peptide and protein analysis were reviewed and evaluated126,127. A review of HPLC methods for the analysis of selected biogenic amines in foods appeared, including methods for extraction and for elimination of interfering compounds128. 

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). 

Overall, microwave-assisted methods are effective approaches in analysis of proteins/peptides, especially when used with other analytical techniques, such as HPLC and MS methods. 

The first comprehensive 2D system was developed in the late 1970s by Erni and Frei, who applied IEX x RPC to the analysis of senna glycosides from plant extracts.61 In the subsequent decades, comprehensive MD-HPLC methods have been further developed, mainly for peptides and proteins,62 3 but also for separation of various natural products such as phenolic and flavone antioxidants64 and carotenoids.65 The theoretical aspects of MD-HPLC techniques have also been further developed.66-68... 

The following sections discuss the application of HPLC to the various methods used in the isolation and analysis of proteins, peptides and amino acids. The development of new stationary phases coupled with instrumentation which allows unattended gradient development has transformed the task of purification. Some of these aspects will also be discussed in this section. 

The reversed-phase HPLC method is a traditional method for the analysis of peptides and proteins. A good choice for the stationary phase could be a short-alkyl reversed-phase (e.g., C4) with wide pores (30 nm). It has been shown that large pore supports give distorted peaks with small collagens and triple helical peptides, resulting in poor resolution. The formation of broad peaks has been ascribed to conformational instability of the separated solutes and slow cis-trans isomerization of the peptide bonds. The best sorbents of those examined were diphenyl or non-porous Cig reversed-phases standard water-acetonitrile gradients were recommended as mobile phases. 

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