Peptide mixture, simple

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. 

MALDI and ESI represent the predominant ionization techniques in mass spectrometry-based proteomics, as recognized by the Nobel Prize in chemistry in 2002. MALDI is mainly used to volatize and ionize simple polypeptide samples for mass spectrometric (MS) analysis at high speed. The analysis of more complex peptide mixtures is usually conducted via ESI mass spectrometry (ESI MS) coupled online with a high-pressure liquid chromatography (HPLC) system to concentrate and separate peptides prior to MS analysis. 

Oxytocin and vasopressin cannot easily be separated from the common aliphatic amino acids on Sephadex G-25 in spite of a considerable difference in molecular size. In a solvent consisting of acetic acid, pyridine, and water (60 15 25 parts by volume) the separation is an easy matter. The explanation is very simple. The gel is not capable of extensive swelling in this solvent and consequently the pores are smaller and the peptides are therefore excluded. This technique has also been used to separate a- and /3-meIanophore-stimulating hormones of the hypophysis (Porath and Schally, 1962). Gel filtration in mixed volatile solvents provides a convenient way to desalt peptide mixtures a problem often difficult to resolve. 

Compared to yeast with about 5800 ORFs which code for proteins, samples from higher organisms can be much more complex and the separation scheme has to be adjusted accordingly. For very simple species as the other extreme, already subcellular fractionation can provide the appropriate pre-separation. For example only one LC-MALDI MS/MS run of a peptide mixture derived from the separated E-coli 50S ribosomal subunit allowed to identify 30 of the 33 expected proteins (Mirgorodskaya et al. 2005a). 

An off-line approach that is simple and useful for peptide/protein sequencing using 5-10 picomoles of material has been demonstrated. Peptide and protein samples were first separated by capillary electrophoresis. Selected peaks were fraction collected and analyzed by both nano-electrospray mass spectrometry and Edman sequencing. A standard peptide mixture, a tryptic-digested protein and intact proteins were used to illustrate this method. Successful fraction collection of each component required reproducible electropherograms, the ability to automatically switch the outlet buffer vessel and the ability to maintain electrophoretic integrity while eluting a peak of interest into a small outlet buffer... 

A simple compartment type of apparatus may be used to separate a peptide mixture into basic, neutral and acidic fractions (Gordon et al., 1941, 1943 Sanger and Tuppy, 1951a). Since the simplification of the mixture is usually more important than the yield of peptides, it is often advisable to repeat the ionophoresis on each fraction, and in this way clear cut fractionations may be obtained with very little overlapping. This method is especially useful for separating the basic peptides. By... 

LC. Whereas MALDI-MS is used to analyze simple peptide mixtures, LC - ESI-MS is more suited to the analysis of complex samples. 

Li, G.-Z., Vissers, J. P. C., Silva, J. C., Golick, D., Gorenstein, M. V. and Geromanos, S. J. Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures. Proteotnics 9 1696-1719,... 

The approach has been employed to determine the amino acid sequence in E, coli alanine t-RNA synthetase. After partial hydrolysis, the peptide mixtures were converted to their methyl esters, N-trifluoroacetylated, reduced with B2D6 and converted to their O-TMS derivatives (9). These compounds possess good GC properties and yield simple, yet informative fragmentations. A variety of derivatives have been explored for sensitive sequence analysis of small peptides and a new permethylation procedure has been described for trifluoroacetylated peptides at the 2 —10 nmol level. Mixtures of these derivatives, ideally of di- to hexa-peptides are readily analyzed by GC/MS 66). 

Peptides are short chains of amino acids. By combining in various ways, two or more of the over 20 amino acids that are made by ail cells, a very large amount of chemical diversity can be generated. Some species of simple organisms such as the bacteria and the cyanobacteria make a number of distinct peptides, each species producing a different mixture. Some of these molecules have been shown to possess biological activity in certain test systems hence these peptides could be considered as NPs. Interestingly, the biosynthesis of these peptides has been compared, as has the biosynthesis of other NPs (see Chapter 5), with nature s version of the chemist s attempts to maximise the production of chemical diversity. ... 

An interesting example of scale-up of peptide synthesis in such low-melting point mixtures derived from eutectic melts has been described [70]. Neat combination of the pure substrates in the complete absence of water/solvent (adjuvant) provided simple heterogeneous systems consisting of the eutectic melts plus an excess of solid substrate (Figure 12.5). 

The photoelectrochemical synthesis of amino acids from simple molecules has also been reported. Low efficiencies were observed in the conversion of mixtures of methane, ammonia and water to several amino acids on platinized TiOz Amino acids and peptides were reported when glucose replaced methane as the carbon source in a parallel experiment Higher quantum efficiencies (20-40%) were observed in the conversion of alpha-keto acids or alpha-hydroxy acids to the corresponding alpha-amino acids Moderate levels of enantiomeric selectivity (optical yields of about 50%) were reported when chiral starting materials were employed. Photoinduced Michael-like reactions were observed when alpha, beta unsaturated acids were used as substrates for the amino acid synthesis... 

---