Peptide standards

Peptides are much more difficult to run by size exclusion chromatography than proteins because their solubilities differ greatly. Their shapes also vary from linear to semidefined. For a three peptide standard, Mant and Hodges... 

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). 

MALDI analysis kits (e.g., Sequazyme peptide mass standards kit from Applied Biosystems) are a convenient way to obtain matrix, peptide standards, and solvents however, these items can easily be purchased independently. Any peptides that span a reasonable proportion of the useful mass range can be used for calibration, provided their accurate molecular weights are known. 

Kuhn E,Wu J, Karl J, et ah Quantification of C-reactive protein in the semm of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards. Proteomics (2004) 4 1-12. 

To reduce the dead volume at the electrospray tip, it was inserted (then glued) into a specially drilled flat-bottom hole, as opposed to a conical-bottom hole [132]. A similar method was used for analysis of various peptide standards and tryptic digests of lectins from Dolichlos biflorus and Pisum sativum [812]. 

While all of these devices used normal zone electrophoresis separation techniques, isoelectric focusing (IEF) methods on microchips have also been interfaced with ESI-MS for high-resolution separations of proteins [37], Figure 5 shows the electropherogram and corresponding mass spectra generated by this device. For on-chip sample preconcentration before separation, a polarityswitching technique was employed to achieve subnanomolar detection limits for many peptide standards [38],... 

Popa, T.V., Mant, C.T., and Hodges, R.S. Ion interaction capillary zone electrophoresis of cationic proteomic peptide standards. 7. Chromatogr. A. 2006, 1111 192-199. 

Since selectivity is a prerequisite for resolution, a temperature increase strongly impacts both selectivity and resolution. The variability of the van t Hoff lines explains the seemingly erratic result of the influence of tanperature on the figures of merit of the method. For example, at lower temperatures, the resolution of model peptides with +1 and +3 net charges improved and worsened, respectively [27]. In chiral IPC, T < 0°C was successfully used to improve enantioresolution [28]. Similarly, lower temperatures provided better resolution in the analysis of a new aminoglycoside antibiotic [29] and for characterization of maize products [30]. Conversely, an increased resolution at 70°C was observed when the ion-pair mechanism was exploited under IPC-capillary zone electrophoresis of cationic proteomic peptide standards [31]. 

NH2-temiinus resulting in a mass of about 30 IdD and 3) an external peptide standard (450 pmol) that was provided dry in an eppendoiftube and that had the same amino acid composition as the unique tryptic peptide insert but whose sequence was randomized. In the case of the two protein samples, 70 pmol of each had been subjected to SDS PAGE and were supplied either as Coomassie Blue stained gel slices or as a section of amido black stained PVDF membrane. In the case of the PVDF samples, an oversize piece of PVDF was included so that a section could be used as a digest control, and in the case of the gel samples, a blank section of gel was included for the same purpose in a separate eppendorf tube. 

A synthetic peptide analogue of the unique insert actually eluted at about 28% CHjCN both from a Vydac C-18 column on the system being tested (data not shown) and from a Zorbax C-18 column on an HPLC system located in a different laboratory (Fig. 1). In the latter instance the peptide insert eluted close to a minor peak eluting at about 54 min in the 28 kD chromatogram. As noted previously, the external peptide standard had the same amino acid composition but a different sequence from that of the unique peptide insert. 

Figure 1. Reverse phase HPLC separation of the external peptide standard (50 pmol, top chromatogram) and an in situ PVDF digest of the 28 kD recombinant (bottom chromatogram). Following SDS PAGE of a mixture of 50 pmol of each protein and blotting onto PVDF, the 28 kD protein was digested with trypsin (3) and subjected to reverse phase HPLC on a Zorbax C18 column (1 x 150 mm) eluted at 37 C at a flow rate of 75 pl/min. The column was equilibrated with 95% buffer A (0.06% TFA) and 5% buffer B (0.055% TFA in CHjCN) and was then brought to 33% and 60% buffer B with linear gradients extending to 63 and 95 min respectively.

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.

For the preparation of cyclic peptides, standard procedures are still the azide and the active ester method. In a variety of bicyclic analogs of somatostatin, azide coupling is applied twice and yields can be improved by catalysis with V-hydroxybenzotriazole. ... 

The Sequazyme C-peptide sequencing kit enables peptide digestion followed by analysis of sequentially truncated peptides using MALDI-TOF-MS. Before digesting and analyzing unknown samples of the peptide (adrenocorticotropic hormone), the activity of the enzyme carboxypeptidase Y (CPY) was checked by dilution experiments with ammonium citrate buffer (pH = 6.1), CPY dilution and the peptide standard angiotensin I. The adrenocorticotropin ACTH (18-39) fragment (0.3 pL) was transferred as a solution of 5 x 10 5M in a 1 1... 

The purity of protected amino acids is especially important for the synthesis of longer peptides. Standard techniques such as melting point determination, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and optical rotation are effective means of characterization. The optical purity can also be evaluated by high-performance liquid chromatography (HPLC) after derivatization with Marfey s reagent [216,217]. The advanced Marfey method refers to analysis by mass spectrometry after derivatization with Marfey s reagent [218-221]. Purification of side-chain protected amino acids by recrystallization is usually sufficient. 

Dissolve the dried peptide calibration standards by adding 125 pL 0.1% TFA to the tube (final concentration will be 4 pmol/pL for each peptide). Mix 1 pL of peptide standard and 9 pL matrix solution and spot 1 pL onto the calibration spots of the AnchorChip target. The final concentration of peptide standard on the target is 400 fmol of each peptide. 

Instrument calibration The instrument is externally calibrated using a linear fit equation with a mixture of six peptide standards (Bruker Daltonics) before data acquisition of the samples (see Subheading 2.4.). 

Custom-made peptide, standard MBP phosphorylated at Thr98, FFBCNIVTPRpTPPPSQGK, (City of Hope, Duarte, CA) is dissolved in PBS at a concentration of 1 mg/mL with 0.02% sodium azide (w/v) and stored at d C. LyophiUzed peptide is stored at -10°C (see Note 5). 

The coupling CE through the sheathless concept is optimally achieved via a nano-ESI source because both operate at uL/min flow rates. In one version, the CE column is connected to the sprayer tip via a microdialysis device [77,78]. In another simple version, a single piece of tapered CE column with a 20-p,m-i.d. tip has been used for separation as well as microspraying [78]. This combination results in high ionization efficiency and low detection limit. The detection limit of 0.1 to 5 finol for peptide standards has been reported. 

---