Sequencing, proteins sample preparation

Most of the molecules of the species to be analysed (analyte) that contribute to the mass spectrum are located in the surface region of the sample. By using adsorption at a solution-solid interface, a uniform layer of the analyte molecules can be made on the surface of a substrate. The substrate is chosen depending on the affinity of the analyte. For example, nitrocellulose is an effective matrix for adsorbing proteins from solution. Hydrophobic substrates, and cationic and anionic substrates have also been used. The sequence for sample preparation is outlined in Figure 3. 

PITC has been used extensively in the sequencing of peptides and proteins and reactions under alkaline conditions with both primary and secondary amino acids. The methods of sample preparation and derivatization follow a stringent procedure which involves many labour-intensive stages. However, the resulting phenylthio-carbamyl-amino acids (PTC-AA s) are very stable, and the timing of the derivatization step is not as critical as when using OPA. 

The Western blot method is often used in the analysis of host cell impurities. It can be used to identify a recurring impurity. O Keefe et al. used a Western blot to identify an E. coli protein impurity in the preparation of the recombinant fibroblast growth factor (aFGF).29 By using specific antisera to the E. coli host cell proteins, they were able to isolate the impurity and determine its N-terminus amino acid sequence to confirm its identity. Antibodies could be used to determine the concentration of this impurity in sample preparations. 

Perform enzymatic hydrolysis of protein samples at a series of time points (see Basic Protocol, steps 1 to 5) and prepare a sequence of standards and a blank (see Basic Protocol 1, steps 6 and 7). Scale up all samples, standards, and blanks to 2.5 ml in 13 x 100-mm glass test tubes. 

Protein samples were obtained from Sigma Chemical Co. (St.Louis,MO). Mouse immunoglobulin G was a generous gift of Dr. J. Bailey, City of Hope. Thiohydantoin-amino acid derivatives were prepared according to published procedures and quality-controlled by HP (3). The HP C-terminal chemical sequencing method was developed for automation using a chemistry licensed by HP from the City of Hope, Duarte, CA. 

Fig. 5 shows the representative sequence data of carbonic anhydrase fria ent 5 from the sample prepared by SDS-PAGE/electroblotting approach. A major sequence of PALKPLALVYGEATS...(starting from residue 41 of the protein) could be identified without difficulty. Another sequence of LKFR IT.NFNAEGEPE... was also found in this sample. 

Many of the more complex sample preparation techniques are used for analysis of biological samples. Liquid-liquid (16) or solid-phase extraction (17) are often used to isolate the compound from the sample, and sometimes it is necessary to apply both techniques in sequence to achieve adequate clean-up (18). Some plasma samples can be analyzed after a quick treatment to remove the protein (67). 

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