Sequencing microsequencing

It should be pointed out that FAB, MALDI, and ESI can be used to provide ions for peptide mass maps or for microsequencing and that any kind of ion analyzer can support searches based only on molecular masses. Fragment or sequence ions are provided by instruments that can both select precursor ions and record their fragmentation. Such mass spectrometers include ion traps, Fourier transform ion cyclotron resonance, tandem quadrupole, tandem magnetic sector, several configurations of time-of-flight (TOF) analyzers, and hybrid systems such as quadrupole-TOF and ion trap-TOF analyzers. 

Peptide microsequencing has proved to be a powerful approach to identify protein components of mixtures,53-55,61 and has been applied to extracted proteins as a method to identify biological threats.56,83-85 Commercial and internet accessible search engines and databases provide adequate bioinformatics support to use sequence information produced by the mass spectrometer (e.g., http //www.matrixscience.com http //prospector.ucsf.edu/ ucsfhtml4.0/mstagfd.htm http //prowl.rockefeller.edu/). 

Bhown, A.S., J.E. Mole, and J.C. Bennett, improved procedure for high-sensitivity microsequencing use of aminoethyl amino-propyl glass beads in the Beckman sequencer and the ultrasphere ODS column for PTH amino acid identification. Anal Biochem, 1981.110(2) 355-9. 

Microsequencers permit sequence analysis on minute amounts of protein. Microsequencing can be used in conjunction with two-dimensional electrophoretic separations of proteins such as that shown in Box 3-C. The proteins in the polyacrylamide gel are electropho-retically transferred onto a porous sheet (membrane) of an inert material such as polyvinyl difluoride.249-251 After staining, a selected spot is cut out and placed into the sequencer. To avoid the problems associated with blocked N termini, the protein may be treated with proteases on the membrane and the resulting peptide fragments may then be separated on a narrow-bore HPLC column and sequenced.240... 

Both Parts I and II have been completely rewritten and reflect the many advances in biochemistry-molecular biology theory and techniques. Especially noteworthy have been the technical advances in chromatography (perfusion, FPLC, bioaffinity), electrophoresis (pulsed gel, capillary, nucleic acid sequencing), spectrophotometry (nmr, ms, and diode array detectors), and molecular biology (microsequencing of proteins and nucleic acids, blotting, restriction enzymes). 

Acrylamide (20%) with 0.5% bisacrylamide is appropriate for the separating gel. PVDF blots are stained with amido black, and all clearly visible bands are excised for direct microsequencing on the membrane, using an Applied Biosystems 476A or 494 sequencer (37)... 

Speicher, D.W. 1989. Microsequencing with PVDF membranes Efficient electroblotting, direct protein adsorption and sequencer program modifications. In Techniques in Protein Chemistry (T. Hugli, ed.) pp. 24-35, Academic Press, San Diego. 

The structure of peptides containing 20 eukaryotic natural amino acids is now routinely determined by the use of automatic protein microsequencer, which uses Edman chemistry to convert each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivative. The formed PTH-amino acids can be identified by their retention times on HPLC systems by comparison with reference standards derived from the 20 natural amino acids. For an OBOC peptide library composed of natural amino acids, the sequencing protocols of the automatic sequencer are well developed and standardized. However, structure determination of peptides composed of unnatural a-amino acids requires modification of the standard sequencing program.32 For peptides composed of non-a-amino acids, one can use an encoding strategy or mass spectrometry if a cleavable linker is employed. In this chapter, we shall focus on the new sequencing method we have developed for unnatural a-amino acids. 

Celis, J. E. Rasmussen, H. H. Leffers, H. Madsen, P. Honore, B. Gesser, B. Dejgaard, K. Vandekerckhove, J. 1991. Human cellular protein patterns and their link to genome DNA sequence data usefulness of two-dimensional gel electrophoresis and microsequencing. FASEB J., 5,2200-2208. 

The elucidation of the primary structures of proctolin (22) and AKH (2Q), signaled the beginning of the structural identification of insect neuropeptides. These relatively small peptides (pentapeptide proctolin and decapeptide AKH) remained the only known insect peptide sequences for a number of years until, in the early 1980 s, accumulated technical advances in peptide isolation and microsequencing techniques facilitated the analysis of insect peptides (8-12 ... 

For the synthetic library method that uses the affinity chromatography selection approach, the bound peptides can be eluted and microsequenced by Edman degradation. Concurrent microsequencing of the retrieved peptide mixture can be performed rather than sequencing individual peptides. Sequeuce motifs then can be defined in a fast and efficient way. However, the amino acid sequence obtained wiU be the result of the summation of the peptide mixture. Uuless a predomiuaut, distinct motif and an alignment of one or more of the critical residues exists within the peptide sequence of the library (e.g., with a fixed residue at a specific position), the result could be very difficult if not impossible to interpret. 

M.W. Hunkapiller and L.E. Hood. 1983. Protein sequence analysis Automated microsequencing Science 219 650-659. (PubMed)... 

While the existence of corticotropin-releasing factor (CRF) was first proposed in 1955 it took 26 years to develop the isolation (RP-HPLC) and sequencing tools (microsequencing) that led to its characterization in 1981.P l First isolated from ovine hypothalanni,P l CRF was subsequently characterized from rat hypothalami P l the identical structure was deduced for human CRF (hCRF) on the basis of the cDNA sequence of the hCRF precursor gene.P CRF plays a major role in the maintenance or restoration of homeostasis by regulating the activity of the hypothalamic-pituitary-adrenal (HPA) axis.b°l Its first synthesis stretched the limits of SPPS capabilities and of preparative HPLC.P l... 

A miniaturized protein and peptide microsequencer consisting of either a fused silica capillary reactor or a microreactor made of Teflon is described. The performance of the miniaturized sequencer was evaluated by sequencing 33 and 27 picomoles of myoglobin that were covalently attached to Sequelon-DITC. The products generated by the sequencer were analyzed using capillary electrophoresis with thermo-optical absorbance detection. This CE system provides reproducible migration time (< 0.4% of RSD) and detection limits of less than 4 fmol. 

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