Gas phase sequencer

Increase in sensitivity and efficiency of analysis in structural studies of enzymes with a gas phase sequencer have made it possible to determine the primary structure in a shorter period of time with a small amount of enzyme at the picomole or even femtomole level. In addition, thanks to the DNA sequencing technique the number of enzymes (or proteins) whose amino acid sequences are registered in a data base file has expanded explosively. The introduction of mass spectrometry on the primary structure determination of protein has stimulated the search for a new methodology other than Edman chemistry. 

Today s predominance of the gas phase sequencer for sequence analysis is partly due to the advancement of PTH-amino acid analysis on HPLC, in which the analysis takes only 10 min to complete using reversed-phase column.70-72 Thus, a gas phase sequencer with an on-line PTH-analyzer can perform a single Edman degradation cycle within one hour. With the use of a microbore column and a data station which handles various data manipulations, a few pmol of PTH-amino acid can be quantitatively analyzed. Although the minimum detection level of amino acid derivatives has improved significantly in the last 10 years (103-104-fold enhancement), further improvement is expected with the use of a fluorescent compound, i.e., fluorescein isothiocyanate (FITC), and the laser detection technique. 

The primary structure of AspAT from Thermophilic bacillus was determined from cDNA sequence.24 Sequence information of an N-terminal portion of the native enzyme and 19 tryptic peptide fragments, recovered from HPLC, was obtained from gas phase sequencer analyses. Based on such sequence information, four oligonucleotide probes were prepared. cDNA encoding AspAT was cloned by screening restriction enzyme fragments from genomic cDNA of Thermophilic bacillus species YM-2. Amino acid sequence of Thermophilic bacillus AspAT deduced from cDNA was confirmed by the sequences made available by gas phase sequencer analysis. 

Perisulfakinin, PSK, was isolated by chance during the investigation of cardioacceleratory-hypertrehalosaemic peptides of P. americana that resulted in the structural characterization of corazonin (5). Amino acid analysis and gas-phase sequencing of an... 

Initial sequencing attempts with HPLC-purified Acheta PDCF indicated a blocked amino-terminus. It was deblocked by pyroglutamyl amino peptidase and subjected to gas-phase sequencing. The resulting data, along with spectral analysis, indicated that the purified peptide has the sequence pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp-amide. This was also the deduced sequence for Gryllus AKH (43). The synthetic peptide. 

Early crystallographic studies of TMADH provided data from two derivatives at 6 resolution that revealed the domain structure and certain elements of secondary structure (Lim et al., 1982 Lim et al., 1984). Higher resolution data at 2.4 resolution have been collected and the structure solved by the multiple isomorphous replacement method with anomolous scattering (Lim et al., 1986). Analysis of the diffraction pattern lead to the identification of ADP as the third cofactor in TMADH. At the time the 2.4 data set was analysed, there was no sequence information available for TMADH (Lim et al., 1986), except for a 12 residue peptide which contained the covalently bound flavin (Kenney et al., 1978). Gas-phase sequencing of isolated peptides initially provided 80% of the primary sequence of... 

Determination of the Disulfide Bonds of Human Macrophage Chemoattractant Protein-1 Using a Gas Phase Sequencer... 

For gas-phase sequencing, the sample is immobilised onto a chemically inert glass frit, often by using a carrier material such as polybrene. Reagents are carried in a gaseous stream of argon and delivered to the glass frit in minute... 

Several gas-phase sequencing techniques have attained success all rely on the generation and mass analysis of sequence-specific fragment ions [38-40], These methods have the advantage of simplicity and are potentially faster than solution-phase methods. 

Isolation of photoaffinity labeled peptides Photoaffinity labeled LSu tryptic peptides were isolated by a procedure involving ion-exclusion, ion-exchange, and reverse-phase HPLC (Salvucci, M.E., Kim, H. and Haley B.E., unpublished). Peptides were subjected to sequence analysis using a gas-phase sequencer (Applied Biosystem 470) and their positions in the intact enzyme were determined by comparison to the known sequence (6,7). Photoaffinity labeled SSu peptides were isolated from spinach Rubisco after separation of the subunits on Sephadex G-75. 

The newest version of the gas-phase sequencer is the so-called pulsed liquid sequencer. Microvalving technology, designed in the original machine, is used... 

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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