HPLC peptide

The sample to be analyzed is introduced to the ESI source by means of a flow stream from an HPLC instrument. The sample flows through a stainless-steel needle and then, sprays out in the form of a mist whose droplets hold peptide ions and mobile phase of HPLC. Peptide ions are separated from the mobile phase and subsequently, transferred into a mass analyzer either by a heated capillary or a curtain of nitrogen gas. Desolvation process can be carried out by a vacuum system. 

Fig. 5 HPLC peptide patterns of fraction IV beef, soy, chicken, and pork.

The TH peptide was purified initially by preparative RP-HPLC on a Rainin AutoPrep System with a Vydac 218TP152022 C18 column (15-20-pm particle size, 300-A pore size, 250 x 22 mm) at a flow rate of 5.0 mL -min The elution gradient was 0-100% B in lOOmin, where A was 0.1% TFA in H20 and B was 0.1% TFA in MeCN, with detection at 229 nm. 1-min fractions were collected, with 10 pL from each fraction used for analytical RP-HPLC. Selected fractions were further purified by semi-preparative RP-HPLC on the same system equipped with a Vydac 219TP510 diphenyl column (5-pm particle size, 300-A pore size, 250 x 10mm) at a flow rate of 2.0mL-min The elution gradient was 0-70% B in 70min, where A was 0.1% TFA in H20 and B was 0.1% TFA in MeCN, with detection at 229 nm. 30-s fractions were collected, with 10 pL from each fraction used for RP-HPLC peptide analyses.1851... 

Peptide mapping by "rapid" HPLC -Peptide mapping by CZE... 

Bizanek R, Manes JD, Fujinari E, Chemiluminescent nitrogen detection as a new technique for purity assessment of synthetic peptides separated by reversed-phase HPLC, Peptide. Res., 9(l) 40 44, 1996. 

Protein Sequencing, Peptide Synthesis, Amino Add Analysis and Mass Spectrometry - Methods for protein modification, proteolysis, RP-HPLC peptide purification and automated Edman degradation are well documented (7,10,14) as are methods for FMOC synthesis and myristylation of synthetic peptides (15) and amino acid analysis (16). 

Comparative HPLC peptide maps of transferrin and myoglobin were used to evaluate relative yields of tryptic peptides from different types of blotting membranes. To ensure identical transfer conditions, replicate lanes from a single gel were transferred to side-by-side strips of nitrocellulose, Immobilon P PVDF and Trans-Blot PVDF as described in Methods. As shown in Fig. 1, our results confirm the observations of Fernandez et al. (15) that optimal... 

Figure 1. HPLC peptide map comparisons (215 nm) using different membrane types. In situ tryptic digestion of replicate apomyoglobin bands (ICX) pmols loaded/lane) electroblotted from a single gel onto 2—Nitrocellulose, 2—Immobilon P, J—Trans-Blot PVDF. B—peaks in a trypsin/buffer control chromatogram. P—PVP-40 peak which is variable from run to run, but more prominent on Trans-Blot membranes.

Figure 2. Reversed Phase Chromatography of HPLC Peptide Standard Overlay of Before and After Microcon-SCX. Starting peptide mixture containing 45 pg in 250 pi of standard or eluted Microcon-SCX. Separation was performed by an Amicon, C18-300-10sp, (4.6 X 250 mm) using a 4 min hold at 15 % ACN, 0.25 % TFA in DIW followed by a linear gradient in 20 min from 15 % ACN to 33 % ACN at 1 ml/min. Approximately 80 % recovery of each peptide was determined by peak area integration ratios.

High-Pressure Liquid Chromatography (HPLC) Peptide Assay... 

Stone, K.L., M.B. LoPresti, J.M. Crawford, R. DeAngelis and K.R. Williams. Enzymatic digestion of proteins and HPLC peptide isolation. In A Practical Guide to Protein and Peptide Purification for Microsequencing, edited by P.T. Matsudaira, San Diego, CA, Academic Press, pp. 31-47, 1989. 

Parker, X, et al. (1986). New Hydrophilicity Scale Derived from HPLC Peptide Retention Data Correlation of Predicted Surface Residues with Antigenicity and X-ray Derived Accessible Sites, Biochemistry 25 5425-5432. 

Detailed descriptions of proteolysis, HPLC peptide mapping, and sequencing of peptide fragments to identify the sites of covalent linkage within the PC-cyt f adduct are being published separately. These studies have shown that there are two sites at which PC is covalently linked to cyt f (Fig. 2). 

Any commercially available or homemade gel electrophoresis apparatus can be used for the elution-concentration system as long as the dimensions do not significantly vary. Electroblotting is carried out in a Bio-Rad Transblot cell or MiniTrans blot. For details on protein sequencing and HPLC peptide separation, reference is made to the article on amino-terminal protein sequence analysis by Heinz Nika and Ruedi Aebersold in this volume. The scalpels (No. 11) were from Paragon and the long needles (Cat. No. V2A 1415 LL-10) from Acufirm. The power supplies were from Pharmacia-LKB (EPS 500/400). 

UV-absorbing compounds are eluted from the gel together with peptides and this may complicate consecutive HPLC peptide separation therefore, it is advisable to use a blank sample too. 

Fig. 9 Localization of the site of PEGylation by C4-HPLC peptide mapping following digestion with endoproteinase Lys-C to release peptide 1-19. Left unmodified IFN-/3-la. Right PEGylated IV N-p-la. Arrowheads (pointing to peak APS) mark the elution position of the peptide that has disappeared in the PEGylated protein, right panel. Reproduced from [55]...

---