Micro quantitative Edman manual sequencing

A manual Edman technique is described which allows sequential quantitative determination of from 3 to 10 amino terminal residues on quantities of peptides or proteins down to one nanomole. This is achieved by a fast, efficient method of obtaining the anilinothiazolinone or phenylthiohydantoin amino acid, and quantitating by either back hydrolysis and amino acid analysis or by a new, rapid, high resolution, quasi-isocratic, high-pressure liquid chromatographic procedure. The overall method has been extensively tested successfully on both peptides and proteins of known and unknown amino-terminal sequence and the results included here. In addition, a wide variety of applications relevant to primary structure analysis such as sequencing blocked polypeptides, use of denaturing agents as coupling buffers, reduction of protein or peptide losses on consecutive sequencing and peptide mixture analysis are all incorporated in the methodology outlined.     

Rapid and reliable peptide de novo sequencing facilitated by microfluidic chip-based Edman degradation

This paper expands the application of the newly developed highly sensitive microfluidic chip-based Edman degradation system. Comparison between the MS/MS spectra of a native peptide and its N-terminus truncated counterpart after carrying out one cycle of Edman degradation in a microfluidic chip can not only provide N-terminal residue information, but also facilitate the identification of different series of fragment ions. Manual peptide sequencing is more feasible and rapid using this method as demonstrated with three peptide examples including one neuropeptide. Furthermore, two cycles of Edman degradation allow the determination of the exact value of b 2 ion of the intact peptide, which can serve as an internal calibrant to increase the mass accuracy of the MS/MS spectrum.     

ABRF ESRG 2005 study: identification of seven modified amino acids by Edman sequencing.

Identification of modified amino acids can be a challenging part for Edman degradation sequence analysis, largely because they are not included among the commonly used phenylthiohydantion amino acid standards. Yet many can have unique retention times and can be assigned by an experienced researcher or through the use of a guide showing their typical chromatography characteristics. The Edman Sequencing Research Group (ESRG) 2005 study is a continuation of the 2004 study, in which the participating laboratories were provided a synthetic peptide and asked to identify the modified amino acids present in the sequence. The study sample provided an opportunity to sequence a peptide containing a variety of modified amino acids and note their retention times relative to the common amino acids. It also allowed the ESRG to compile the chromatographic properties and intensities from multiple instruments and tabulate an average elution position for these modified amino acids on commonly used instruments. Participating laboratories were given 2000 pmoles of a synthetic peptide, 18 amino acids long, containing the following modified amino acids: dimethyl- and trimethyl-lysine, 3-methyl-histidine, N-carbamyl-lysine, cystine, N-methyl-alanine, and isoaspartic acid. The modified amino acids were interspersed with standard amino acids to help in the assessment of initial and repetitive yields. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the ESRG Web site. The ABRF ESRG 2005 sample is the seventeenth in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.     

Application of high-performance liquid chromatographic peptide purification to protein microsequencing by solid-phase Edman degradation

Peptide purification via high-performance liquid chromatography (HPLC) and solid-phase sequencing were integrated to form a system allowing the determination of complete sequence information on a microscale without the use of radiolabels or modified phenylisothiocyanate. Mixtures of peptides (500 pmol to 10 nmol) resulting from proteolytic digestion or chemical cleavage were applied directly to reverse-phase columns. The columns, equilibrated in either 10 mm KP_i or 0.05% trifluoroacetic acid, were then developed using acetonitrile gradients. Eluates were monitored nondestructively by direct ultraviolet detection at both 214 and 254 nm. Each peak was collected as a discrete fraction, and purity was assessed by amino acid analysis prior to covalent attachment to a solid support for sequence analysis. Activation of the peptide carboxyl terminus via a water soluble carbonyldiimide was the solid-phase coupling method used 90% of the time. Coupling yields averaged 52% of starting material. Sequence analysis was performed in the range 100 pmol to 4 nmol of coupled peptide. Phenylthiohydantoin-amino acids were identified by reverse-phase HPLC using ultraviolet detection.     

Assignment of the disulfide bonds of huwentoxin-II by Edman degradation sequencing and stepwise thiol modification.

A novel strategy combining Edman degradation and thiol modification was developed to assign the three disulfides of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of the spider Selenocosmia huwena. Phenylthiohydantoin (Pth) derivatives of Cys and the elimination product, dehydroalanine (DeltaSer), can be observed in the Cys cycles during Edman degradation of native HWTX-II. The appearance of two products indicates that the disulfides of HWTX-II were split and that the free thiol group of the second half cystine has been generated. Information about the nature of the disulfide bridges of HWTX-II could be obtained from the sequencing signal if the nascent thiols were modified stepwise by 4-vinylpyridine. Using this method the disulfide bridges of HWTX-II were assigned as Cys4-Cys18, Cys8-Cys29 and Cys23-Cys34, which is different from that seen in HWTX-I, a neurotoxic peptide from the same spider. Using this strategy, one can assign the disulfide bonds of small proteins by sequencing and modification n - 1 times, where n is the number of disulfide bonds in the protein. The above assignment of the disulfide bonds of HWTX-II was confirmed by MALDI-TOF MS of tryptic fragments of HWTX-II. Some disulfide interchanging during proteolysis was observed by monitoring the kinetics of proteolysis of HWTX-II by MALDI-TOF MS.     

A new hybrid resin for stepwise screening of peptide libraries combined with single bead Edman sequencing

A library system was developed for the discovery of bioactive peptides. Library synthesis and peptide sequencing was performed on a solid support while the screening for bioactivity was done with peptides in solution. The peptides were synthesized by split and mix, one-bead–one-peptide library synthesis, using a Tentagel S-NH₂ solid support with a loading of approximately 100 pmol/bead. The major part of the peptide was connected to the support by a single acid-labile linker and a minor part of the peptide was acid-stabile attached to the polymer. The percentage of acid-stabile attached peptides could easily be controlled during modification of the amino functionalities of the resin at the start of the process. The cleavage rate of the acid-labile attached peptide from the resin depends on the composition of the cleavage mixture. When cleavage conditions were carefully controlled, a three-step partial cleavage protocol allowed for convergent bioactivity screening on peptide libraries using only one type of acid-labile linker. The partial cleavage and convergent screening procedure was repeated three times, after which the bead containing the bioactive peptide was sequenced. As such a bead still contained acid-stabile attached peptide, the Edman sequencing was straightforward and repetitive yields were excellent because the immobilized peptide was not washed out. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

SPR biosensing coupled to a digital microfluidic microstreaming system

This article reports on a proof-of-concept system composed of a droplet based surface plasmon resonance (SPR) system coupled to a surface acoustic wave (SAW) microfluidic plateform. It is now well established that surface based binding analyses such as SPR are highly influenced by the transport of analyte to the sensing surface. Further, obtaining reliable equilibrium in flow cells to realize quantification studies is not straightforward. An original solution compared to generally used pressure driven flows is then proposed to favourably cope with these issues. Efficiency of SAW microstreaming coupled to SPR biosensing is considered, in order to improve the accuracy of kinetic parameter estimation in mass transport limited regime and to realize reliable quantification studies. First, the droplet based SPR technique and its advantages are presented. Then, the integration of the microstreaming on the system is discussed. Streptavidin binding is then monitored in static mode and under SAW streaming mode.     

Identification of unusual amino acids in peptides using automated sequential Edman degradation coupled to direct detection by electrospray-ionization mass spectrometry

The determination of the primary structure of peptides and proteins is routine in many laboratories; however, many of the obtained sequences are incomplete or can be misinterpreted when the samples contain unusual amino acids. Here we report the development of an automated peptide sequenator coupled to an electrospray-ionization (ESI) mass spectrometer (MS) that, in conjunction with minor modifications to the sequencing conditions and, in some cases, prior derivatization of amino acids, allows the detection of the phenylthiohydantoin (PTH) derivatives of a number of unusual amino acids. Using the coupled sequenator-ESI-MS system we were able to determine the complete sequence of the lantibiotic gallidermin, a partial sequence of the calcium-dependent peptide antibiotic CDA2 as well as the pool sequence of a mixture of synthetic peptides containing nonproteinogenic amino acids. In addition to the 20 proteinogenic amino acids, the procedure was able to detect PTH derivatives of hydroxyphenylglycine, 2,3-didehydroasparagine, 3-methylglutamic acid, oxytryptophan, ornithine, N-methylglycine, dihydroxyphenylalanine, and alpha-aminoisobutyric acid. Similarly, after a simple derivatization procedure, we were also able to correctly identify educts of 2,3-didehydroalanine, 2,3-didehydrobutyrine, lanthionine, and 3-methyllanthionine.     

VSQual: a visual system to assist DNA sequencing quality control

A lack of pliant software tools that support small- to medium-scale DNA sequencing efforts is a major hindrance for recording and using laboratory workflow information to monitor the overall quality of data production. Here we describe VSQual, a set of Perl programs intended to provide simple and powerful tools to check several quality features of the sequencing data generated by automated DNA sequencing machines. The core program of VSQual is a flexible Perl-based pipeline, designed to be accessible and useful for both programmers and non-programmers. This pipeline directs the processing steps and can be easily customized for laboratory needs. Basically, the raw DNA sequencing trace files are processed by Phred and Cross_match, then the outputs are parsed, reformatted into Web-based graphical reports, and added to a Web site structure. The result is a set of real time sequencing reports easily accessible and understood by common laboratory people. These reports facilitate the monitoring of DNA sequencing as well as the management of laboratory workflow, significantly reducing operational costs and ensuring high quality and scientifically reliable results.     

Application of a coupled enzyme assay to characterize nicotinamide riboside kinases

The recently identified nicotinamide riboside kinases (Nrks) constitute a distinct pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. Here we present the combination of an established optical adenosine triphosphatase (ATPase) test, the pyruvate kinase/lactate dehydrogenase system, with the Nrk-catalyzed reaction to determine kinetic properties of these enzymes, in particular affinities for ATP. The assay allows variation of both nucleoside and phosphate donor substrates, thereby providing major advantages for the characterization of these enzymes. We confirm previously established kinetic parameters and identify differences in substrate selectivity between the two human Nrk isoforms. The proposed assay is inexpensive and may be applied for high-throughput screening.     

Use of o-phthalaldehyde to reduce background during automated Edman degradation

Background generated during the cleavage step of the Edman degradation is a major obstacle to extended automated amino acid sequencing. It was demonstrated recently by A. S. Bhown, J. C. Bennett, P. H. Morgan, and J. E. Mole (1981, Anal. Biochem. 112, 158-162) that introduction of fluorescamine into the spinning cup reduced background by blocking primary amino groups at cycles where a proline residue is at the exposed amino terminus. A convenient blocking reaction program using o-phthalaldehyde which can be intercalated into the sequencing format of an automated Beckman liquid-phase sequencer is presented. The advantages of o-phthalaldehyde in blocking of primary amines when proline is amino terminal arise from its stability in aqueous solution and the ease of programmed metering of delivery to the sequencer spinning cup. The blocking reaction proved successful not only in extending sequence analyses but also in the elimination of unwanted sequences in selected peptide mixtures without the necessity of purification of the target peptide.     

Characterization and analysis of posttranslational modifications of the human large cytoplasmic ribosomal subunit proteins by mass spectrometry and Edman sequencing

The 60S ribosomal proteins were isolated from ribosomes of human placenta and separated by reversed phase HPLC. The fractions obtained were subjected to trypsin and Glu-C digestion and analyzed by mass fingerprinting (MALDI-TOF), MS/MS (ESI), and Edman sequencing. Forty-six large subunit proteins were found, 22 of which showed masses in accordance with the SwissProt database (June 2002) masses (proteins L6, L7, L9, L13, L15, L17, L18, L21, L22, L24, L26, L27, L30, L32, L34, L35, L36, L37, L37A, L38, L39, L41). Eleven (proteins L7, L10A, L11, L12, L13A, L23, L23A, L27A, L28, L29, and P0) resulted in mass changes that are consistent with N-terminal loss of methionine, acetylation, internal methylation, or hydroxylation. A loss of methionine without acetylation was found for protein L8 and L17. For nine proteins (L3, L4, L5, L7A, L10, L14, L19, L31, and L40), the molecular masses could not be determined. Proteins P1 and protein L3-like were not identified by the methods applied.