Cytochrome tryptic digests

Bushey, M.M., Jorgenson, J.W. (1990a). A comparison of tryptic digests of bovine and equine cytochrome c by comprehensive reversed-phase HPLC-CE. J. Microcolumn Separations 2, 293-299. 

Wahl, J.H., Gale, D.C., Smith, R.D. (1994). Sheathless capillary electrophoresis-electrospray ionization mass spectrometry using 10 pm I.D. capillaries analyses of tryptic digests of cytochrome c. J. Chromatogr. 659, 217-222. 

Separation of a tryptic digest of cytochrome c Analysis of neuropeptides... 

Another approach has been the preparation of fragments of cytochrome c, in which the heme group is bound to a portion of the protein, either by tryptic digestion or cyanogen bromide cleavage. 

Figure 8.18 ESI mass spectrum of cytochrome c (43.8 fmol) tryptic digest obtained by summing over the range 18.5 to 19.3 min [148].

Two-dimensional liquid phase separation was conducted on-chip. A MEKC/CE was first reported. Here, the first dimensional MEKC separation was followed by a second-dimensional CE separation to separate the tryptic digests of cytochrome c, 5-lactalbumin, and ribonuclease A. However, the CE and MECC separations are not completely orthogonal (or uncorrelated), limiting the resolving power of this method [666]. 

A single microchannel has been used for sequential MS analysis of multiple samples. In this case, no sample cross-contamination has been reported. For instance, no sample crossover was observed for 10 iM cytochrome c versus 10 iM ubiquitin [943], and tryptic digests of [3-lac, CA, and BSA [776]. In another report, sequential infusion of tryptic digests of CA (290 nM) and BSA (130 nM) into ESI-ITMS was achieved using EOF. No cross-contamination resulted when a central flow of buffer confined the other samples in the reservoir and channel by precise voltage control [801]. 

Transfer capillary integrated nebulizer, glass chip Pneumatic nebulization(150 nL/min) ESI-ITMS Angiotensin peptides (20 (Xg/mL) cytochrome c tryptic digest Trypsin ITP-CE 802... 

Inserted capillary tip (no glue), glass chip Pressure flow 100 nL/min ESI-ITMS (LCQ) Tryptic digest of cytochrome c myoglobin, (3-lactoglobulin A B, BSA Trypsin CE 813... 

Tryptic digest of bovine hemoglobin (4 pM) cytochrome c (0.8 pM) Human hemoglobin (normal and sickle cell, 0.24 pM) Mixed off-chip/on-chip tryptic digestion Nil 807... 

Fig. 8.24. Separation of a tryptic cytochrome C digest by pressure assisted gradient CEC. Column, 60 x 0.18 mm i.d. packed with 3 pm Vydac Cl8 eluents, (A) 0.07% trifluoroacetic acid in water, (B) 0.07% trifluoroacetic acid in acetonitrile gradient elution with 0-50% B in 20 min applied pressure, 9 MPa in (a), 5 MPa in (b) 7 MPa in (c) applied voltage, 0 kV in (a), 1 kV in (b), 0.6 kV in (c) Detection, nanospray ESI-MS, m/z 200-1500, 10 spectra/s sample, tryptic digest of 8 pmol bovine cytochrome C. (Reprinted with permission from ref. [45], Copyright [1997] American Chemical Society).

Cytochrome c, bovine, tryptic digest, chicken albumin, tryptic digest Vydac Cl8, 3 pm Acetonitrile-0.07% TFA in water (gradient from 0 100 to 40 60) 60 mm x 180 pm i.d. 99... 

Recently, rapid separation of peptide and proteins by CEC was obtained by increasing the separation temperature [52], A tryptic digest of cytochrome c was obtained in less than 5 min at 55°C. Arrhenius plots of logarithmic p against the reciprocal absolute temperature for both peptides and proteins also yielded straight lines. 

N-alkyl silica-based stationary phases typically employed in HPLC separation of peptides and proteins have been investigated to a large extent in CEC mode. In most cases, gradient elution was required to optimize the resolution and analysis time. Tryptic digests maps of cytochrome c by pressurized CEC were better resolved than by pHPLC [93], The experiments were conducted at low pH in the presence of trifluoroacetic acid (TFA) to prevent tailing of basic peptides, on a column packed with 1,5-pm C18 silica particles. It has been... 

Figure 20 Tryptic digest of cytochrome c labeled with NDA 100-pm-i.d. negative lauryl column buffer-25/75, ACN/25 mM PB, pH 6.9, 5-s injection at 66 V/cm, run at 167 V/cm. (Reprinted from Ref. 96, with permission.)...

Figure 3-23. RP separation of bovine cytochrome c tryptic digest at flow rates (A) l.OmL/min with 60-min gradient and (B) lO.OmL/min with 6-min gradient. (Reprinted...

Figure 1. Separations of Cytochrome c tiyptic digests. (A) 5 xl of the tryptic digest (0.86mg/ml) was injected to give approximately 360 pmol separated on the 3.9mm ED column. Peaks are labeled according to Reference 4. (B) A 5pl aliquot of the 10-fold diluted digest (ca. 36 pmol) was separated on the 2mm ID column and this same sample was derivatized with AQC and 50pl injected. Other conditions are described in the Experimental Section.

Figure 4. Separation of 860 finol of Cytochrome c tryptic digest using (A) a 3.9mm ID column with a Spl flow cell, (B) a 2mm column with the Spl flow cell, (C) a 3.9mm column with the 16pl flow cell, and (D) a 2mm column with the 16pl flow cell. All other conditions are described in the experimental section. Peaks marked with an asterisk ( ) are found in derivatization blanks. Other peaks are components in the digest that are derivatized.

Figures. High sensitivity analysis of Cytochrome c tryptic digests. Samples were analyzed on the 2mm ID column using a 5 1 flow cell. Amounts injected were (A) 860 fmol, (B) 172 finol and (C) a derivatization blank with reagent amount equivalent to the analysis in (A) and five times the reagent injected in (B). Major peaks present in the blank are labeled with an asterisk ( ).

Figure 1. Chromatogram of Cytochrome C tryptic digestion mixture. Lettered peaks were analyzed with the use of both PB LC/FT-IR spectrometry and LC/MS.

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