Peptide mixtures, analysis

COUPLED MULTIDIMENSIONAL CHROMATOGRAPHY AND TANDEM MASS SPECTROMETRY SYSTEMS FOR COMPLEX PEPTIDE MIXTURE ANALYSIS... 

Wu, J. T., Huang, P. Q., Li, M. X., Qian, M. G., and Lubman, D. M. (1997). Open-tubular capillary electrochromatography with an on-line ion trap storage/reflectron time-of-flight mass detector for ultrafast peptide mixture analysis. Anal. Chem. 69, 320-326. 

Hoaglund-Hyzer, C.S. Clemmer, D.E., Ion trap/ion mobility/quadrupole/time-of-flight mass spectrometry for peptide mixture analysis. Anal. Chem. 2001, 73, 177-184. 

Fig. 4 The total ion chromatography (TIC) of the separation of a hyptic digest of chicken ovalhumin with a sample injection amount of 12 pmol corresponding to the original protein. Column length, 6 cm. Conditions 20 min, 0-40% acetonitrile gradient 1000 V apphed voltage with a 40-har supplementary pressure. Source From Open-tuhular capillary electrochromatography with an onhne Ion trap storage/reflectron time-of-flight mass detector for ultrafast peptide mixture analysis, in Anal. Chem.f with permission of the authors and the American Chemical Society.

Nielsen, P. F., and Roepstorff, P. (1989) Suppression effects in peptide mixture analysis by plasma desorption mass spectrometry. Biomed. Environm. Mass Spectrom. 18,131-137. 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

A. J. Tomlinson and S. Naylor, Enhanced performance membrane preconcentration-capillary electiophoiesis-mass spectiometry (mPC-CE-MS) in conjunction with transient isotachophoresis for analysis of peptide mixtures, J. High Resolut. Chromatogr. 18 384-386(1995). 

In 1990, Bushey and Jorgenson developed the first automated system that eoupled HPLC with CZE (19). This orthogonal separation teehnique used differenees in hydrophobieity in the first dimension and moleeular eharge in the seeond dimension for the analysis of peptide mixtures. The LC separation employed a gradient at 20 p.L/min volumetrie flow rate, with a eolumn of 1.0 mm ID. The effluent from the ehromatographie eolumn filled a 10 p.L loop on a eomputer-eontrolled, six-port miero valve. At fixed intervals, the loop material was flushed over the anode end of the CZE eapillary, allowing eleetrokinetie injeetions to be made into the seeond dimension from the first. 

Figure 11.18 Schematic diagram of an in-line SPE unit for CE using (a) polyester wool frits to hold the sorbent, or (b) a paiticle-loaded membrane. Reprinted from Journal of Capillary Electrophoresis, 2, A. J. Tomlinson and S. Naylor, Enhanced performance membrane preconcenti ation-capillary electrophoresis-mass spectiometi y (mPC-CE-MS) in conjunction with ti ansient isotachophoresis for analysis of peptide mixtures, pp 225-233, 1995, with permission from ISC Teclmical Publications Inc.

Figure 5.31 LC-electrospray-MS-MS spectrum of the column eluate at around 22 min in the analysis of the peptide mixture from the tryptic digest of glycoprotein TIME-EA4 from silkworm diapause eggs. Reprinted from Bioorg. Med. Chem., 10, Kurahashi, T., Miyazaki, A., Murakami, Y., Suwan, S., Franz, T., Isobe, M., Tani, M. and Kai, H., Determination of a sugar chain and its linkage site on a glycoprotein TIME-EA4 from silkworm diapause eggs by means of LC-ESI-Q-TOF-MS and MS/MS , 1703-1710, Copyright (2002), with permission from Elsevier Science.

Opiteck, G.J., Jorgenson, J.W., MacNair, J.E., Moseley, M.A., 3rd (1997). Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides. Rapid separation and characterization of protein and peptide mixtures using 1.5 microns diameter non-porous silica in packed capillary liquid chromatography/mass spectrometry. Anal. Chem. 69, 2283-2291. 

The ability to resolve and characterize complicated protein mixtures by the combination of 2DLC and online mass spectrometry permits the combination of sample fractionation/simplification, top-down protein mass information, and bottom-up peptide level studies. In our lab, the simplified fractions generated by 2D(IEX-RP)LC are digested and analyzed using common peptide-level analysis approaches, including peptide mass fingerprinting (Henzel et al., 1993 Mann et al., 1993), matrix-assisted laser desorption/ionization (MALDI) QTOF MS/MS (Millea et al., 2006), and various capillary LC/MS/MS methodologies (e.g., Ducret et al., 1998). 

HPLC fractionation is the first step (dimension) in the two-dimensional analysis of complex peptide mixtures. This step is generally carried out by gradient elution... 

The peptide mixture on the MALDI target can be exposed to a chemical derivatization to confirm the identity of a peptide by the mass shift associated with the sequence-specific derivatization. A large number of possible derivatization reactions can be combined with the MALDI-TOF analysis. Their usefulness depends critically on the kinetics of the derivatization reaction, whether the reaction is complete with small amounts of peptides and whether only one product is generated. A visible MALDI signal can be generated from low atomole of peptide present under the laser beam (Vorm et al., 1994), but these amounts are often not sufficient... 

The primary goal of peptide mapping is the verification of the amino acid sequence deduced from the genetic code of the recombinant protein. The protein backbone gets cleaved by typically two or three different endoproteinases like Lys-C, trypsin, and Glu-C to achieve maps with sequence-overlapping peptide fragments. These peptide mixtures can then be separated by LC or CE and analyzed on-line by MS to obtain sequence information. Often simple mass analysis matches the predicted primary sequence of the protein. However, sometimes mutations can lead to isobaric masses of peptides that can be overseen, if no further sequence analysis like N-terminal Edman sequencing and MS/MS is carried out. 

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