Peptides high sensitivity sequence analysis

The Use of a Volatile N-Terminal Degradation Reagent for Rapid, High-Sensitivity Sequence Analysis of Peptides by Generation of Sequence... 

Electrospray in the mid 1980s revolutionized biological mass spectrometry, in particular in the field of protein and peptide sequence analysis. Electrospray is a concentration-dependent, rather than a mass-dependent process, and maximum sensitivity is achieved at low flow rates with high-concentration, low-volume samples (Griffiths 2000). Joint NMR, x-ray diffraction, electrophoresis, and chromatography techniques with mass spectrometry (MS) techniques would be a trend in the future. 

The sequencing strategy follows the order 1) preparation of pure protein, 2) selective cleavage of peptide bond 3) isolation of cleaved peptide fragments, 4) analysis of primary structure on amino acid sequencer, 5) determination of entire primary structure. Although this order has not changed since our primary structure determination work on AspAT carried out in early 1970, many improvements have been made, e.g. preparation of micro scale amount of sample and introduction of high sensitivity analytical instruments. These improvements have shortened analysis time and lowered the sample amount of required. 

The increased mass accuracy available when the instrument was operated in the reflectron mode was important for the analysis carried out. For example, fractions 4b and 7 or fractions 5 and 8 from C. ermineus appeared to be the same species when measured with the instrument operated in the linear mode. Only in the reflectron mode were we able to reliably distinguish the masses of each species. The high sensitivity of MALDI-TOF is particularly important for the analysis of native peptides such as conotoxins where often the venom of many milkings must be collected to obtain sufficient material for sequence analysis. The increased sensitivity of MALDI over LSIMS is illustrated in the analysis of fraction 5 from C. striatus venom (see Table I). Despite the two orders of magnitude difference in the amount of material consumed in the LSI experiment we did not discern any intact species in fraction 5, whereas the MALDI measurement yielded useful information. However, the comparisons in Tables I and II reveal that some components may be detected by LSIMS but not observed in the MALDI mass spectrum (measured with any of the matrices or sample preparation methods). The contrary is most likely more prevalent, i.e. that a large number of the species detected by MALDI with one or more of the matrices are difficult species to ionize with LSIMS. 

High Sensitivity Peptide Sequence Analysis Using In Situ Proteolysis on High Retention PVDF Membranes and a Biphasic Reaction Column Sequencer... 

C. Evaluation of the HP Sequencer for High Sensitivity Peptide Sequence Analysis... 

R. Aebersold, G.D. Pipes, R.E. Wettenhall, H. Nika, and L.E. Hood. 1990. Covalent attachment of peptides for high sensitivity solid-phase sequence analysis Ana/. Biochem. 187 56-65. (PubMed)... 

The most popular method is automated Edman chemistry (mn on a sequencer), a method which removes one amino acid at a time from the N-terminus, resulting in the sequential liberation of phenylthiohydantoin (PTH) amino acids, which are identified by on-line HPLC analysis. Edman sequanators are completely automated high-sensitivity instrument systems that can routinely detect and identify as little as 0.2 pmol to 1 pmol of amino acid in a given cycle and carry out more than 20 cycles with 1 to 5 pmol of protein. Most peptides of length 3-30 amino acids can be sequenced completely. Although amino... 

The newest gas-phase sequencers also incorporate an automated conversion chamber to facilitate the on-line analysis of the PTH amino acids by microbore LC. All of these features facilitate rapidity and high sensitivity in microsequencing of peptides and proteins and have drastically decreased the amounts of material needed for structure determination to the extent that direct N-terminal sequence analysis of over 20 residues from a 2-D gel is possible. As a result, this methodology has altered the strategy for protein isolation from the tissue in which it was initially detected. 

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