Identification of major histocompatibility complex class II-associated peptides derived from freshly prepared rat Langerhans cells using MALDI-PSD and Edman degradation

The isolation and identification is described of MHC class II-bound peptides derived from Langerhans cells. A combination of preparative micro-HPLC, MALDI-MS, Edman degradation was used for determining the amino acid sequence of MHC-associated peptides. Sample handling was crucial because fractions containing trace amounts of material require immediate storage at -80 degrees C to prevent peptide losses.     

Computer analysis of automated Edman degradation and amino acid analysis data

Computer programs are described that allow facile analysis of data from a protein sequencer and amino acid analyzer. The sequencer program provides automated sequence interpretation while requiring minimal user interaction. The program serves as a powerful aid in deciphering mixture sequences and allows routine monitoring of sequencer performance. The computer program for amino acid analysis data provides the following calculations: mole percent, protein concentration and residues per mole with comparison between theoretical and calculated values. A plot of molecular weight versus deviation from integer values is calculated providing a measure of peptide or protein purity.     

Subfemtomole level protein sequencing by Edman degradation carried out in a microfluidic chip

A novel microfluidic chip based Edman degradation system is developed, in which Edman degradation can be carried out with peptide at a subfemtomole level; combined with MALDI-TOF-MS detection, the identification specificity of gel-separated protein in low abundance has been drastically improved.     

Edman降解中异硫氰酸苯酯新修饰位点的发现和验证

Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和液相色谱-串联质谱（LC-MS/MS）研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。     

Edman Stepwise degradation of polypeptides: a new strategy employing mild basic cleavage conditions

Terminal $\text{N}$-(phenylthiocarbamoyl)peptides, formed under mild basic conditions in the first stage (“coupling”) of the Edman degradation of peptides, are shown to undergo cleavage at the first peptide bond under similar conditions at higher termperatures. The usual products of the Edman degradation are formed (the phenylthiohydantoin of the $\text{N}$-terminal amino acid, and the correspondingly shortened peptide) in one step.     

High-throughput sequence determination of cyclic peptide library members by partial Edman degradation/mass spectrometry

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.     

Reliable sequence determination of ribosome- inactivating proteins by combining electrospray mass spectrometry and Edman degradation

The primary structure of saporin-S9 and MAP-S, two type-1 ribosome-inactivating proteins isolated from the seeds of Saponaria officinalis L. and Mirabilis jalapa, respectively, was determined using a combined approach based on Edman degradation and electrospray ionization mass spectrometry (ESMS). Saporin-S9 has 253 amino acids with a calculated molecular mass of 28,492.99, which is in good agreement with that determined by ESMS (28 495 +/- 2 Da). Unlike other saporins with known primary structure, saporin-S9 contains four histidinyl residues (positions 111, 121, 216 and 248). By comparing the amino acid sequence of saporin-S9 with that of saporin-S6, we found 22 amino acid substitutions (8.7%), 13 of which are conservative and nine non-conservative. The residues known to be involved in the definition of the active site and with RNA base recognition are conserved. The four histidinyl residues and especially Lys for Gln203 contribute to the higher calculated pI value (10.17) of saporin-S9 compared with saporin-S6 (9.98). MAP-S contains 250 amino acid residues with a calculated molecular mass of 27,789.49, in good agreement with that determined by ESMS (27,789 +/- 2). Cys36 and Cys220 form a disulphide bridge and only four amino acid residues are different from the amino acid sequence of MAP, isolated from the roots of the same plant, i.e. Leu34 (Glu), Ile161 (Leu), Asp185 (Glu) and Asp191 (Glu) (in parentheses, the residues present in MAP). The reported approach can provide rapid and reliable sequence screening in the analysis of homologous proteins, including the presence of disulphide bridges.     

Sequencing of sulfonic acid derivatized peptides by electrospray mass spectrometry

We report the application of nanoelectrospray ionization tandem mass spectrometry (nES-MS/MS) and capillary LC/microelectrospray MS/MS (cLC/&mgr;ES-MS/MS) for sequencing sulfonic acid derivatized tryptic peptides. These derivatives were specifically prepared to facilitate low-energy charge-site-initiated fragmentation of C-terminal arginine-containing peptides, and to enhance the selective detection of a single series of y-type fragment ions. Both singly and doubly protonated peptides were analyzed by MS/MS and the results were compared with those from their derivatized counterparts. Model peptides and peptides from tryptic digests of gel-isolated proteins were analyzed. Derivatized singly protonated peptides fragment in the same way by nES-MS/MS as they do by post-source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD-MALDI-MS). They produce fragment ion spectra dominated by y-ions, and the simplified spectra are readily interpreted de novo. Doubly protonated peptides fragment in much the same way as their non-derivatized doubly protonated counterparts. The fragmentation of doubly protonated derivatives is especially useful for sequencing peptides that possess a proline residue near the N-terminus of the molecule. The singly protonated forms of these proline-containing derivatives often show enhanced fragmentation on the N-terminal side of the proline and considerably reduced fragmentation on the C-terminal side. In addition, sulfonic acid derivatization increases the in-source fragmentation of arginine-containing peptides. This could be useful for sequence verification and sequence tagging for use in single stage mass spectrometry. Copyright 2000 John Wiley & Sons, Ltd.     

Micro-scale sequence analysis from the N-terminus of peptides using the fluorogenic Edman reagent 4-N,N-dimethylamino-1-naphthyl isothiocyanate

The fluorogenic Edman reagent 4-N,N-dimethylamino-1-naphthyl isothiocyanate (DNTC) was reacted with amino acids and peptides, cyclized by acid and the liberated 4-N,N-dimethylamino-1-naphthyl thiohydantoin (DNTH) amino acids were then separated and detected by HPLC. The fluorescence intensities of DNTH-amino acids except DNTH-proline and -serine were dramatically increased in the alkaline solution and organic solvent. Thus, the postcolumn reaction with alkaline acetonitrile solution was adopted in HPLC. The polar and aromatic amino acids afforded two DNTH-amino acids on derivatization with DNTC and cyclization with acids. These were suggested to be stereoisomers of DNTH-amino acids. The sequence analysis of 0.5 nmol Leu-enkephalin was achieved by the double coupling method with DNTC and phenyl isothiocyanate followed by the proposed HPLC system.     

Morphology transformation via pH-triggered self-assembly of peptides

Three flexible peptides (P1: (C(17)H(35)CO-NH-GRGDG)(2)KG; P2: (Fmoc-GRGDG)(2)KG; P3: (CH(3)CO-NH-GRGDG)(2)KG) self-assembled to form a variety of morphologically distinct assemblies at different pHs. P1 formed nanofibers at pH 3, then self-assembled into nanospheres with pH up to 6 and further changed to lamellar structures when the pH value was further increased to 10. P2 aggregated into an entwined network structure at pH 3, and then self-assembled into well-defined nanospheres, lamellar structures, and vesicles via adjusting the pH value. However, P3 did not self-assemble into well-ordered nanostructures, presumably due to the absence of a large hydrophobic group. The varying self-assembly behaviors of the peptides at different pHs are attributed to molecular conformational changes. These self-assembled supramolecular materials might contribute to the development of new peptide-based biomaterials.     

Use of Edman degradation sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry in designing substrates for matrix metalloproteinases

The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. We have been designing single-stranded peptides (SSPs) and triple-helical peptides (THPs) as potential discriminatory MMP substrates. Edman degradation sequence and matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analyses of proteolytic activity have been utilized to aid in further substrate design. THP models of the alpha1(I)772-786 sequence from type I collagen were synthesized to examine the triple-helical substrate specificity of MMP family members. Sequence and MALDI-MS analyses were used in conjunction with a fluorometric assay to determine the exact point of cleavage by each MMP. MMP-1 (interstitial collagenase) cleaved the substrates at a single Gly-Ile bond, analogous to the cleavage site in type I collagen. MMP-2 (Mr 72 000 type IV collagenase; gelatinase A) was found to cleave the substrates at two sites, a Gly-Ile bond and a Gly-Gln bond. MMP-3 (stromelysin 1) was found to cleave only one of the substrates after reaction for 48 h. Ultimately, sequence and MALDI-MS analyses allowed us to detect an additional cleavage site for MMP-2 in comparison to MMP-1, while MMP-3 was found to cleave a substrate after an extended time period. The second cleavage site would cause the kinetic parameters for MMP-2 to be overestimated by the fluorometric assay. Further design variations for these substrates need to consider the presence of more stable triple-helical conformation (to eliminate MMP-3 binding) and the removal of Gly-Gln bonds that may be susceptible to MMP-2.     

Microbial degradation of physiologically active peptides by strain B-9

The reaction of some physiologically active peptides with bacterial strain B-9 has been investigated. Bradykinin, β-endorphin, and [Leu(5)]enkephalin were quickly degraded, with half-lives of <5 min. Somatostatin, substance P, and angiotensin I were degraded relatively smoothly, with half-lives of 10 min to 1 h, whereas oxytocin and insulin were slowly degraded, with half-lives of 1 and 4 days, respectively. Vasopressin was barely degraded, with a half-life of >7 days. Linearized vasopressin, prepared by the reductive cleavage of the disulfide bond followed by alkylation with iodoacetamide, was degraded significantly faster than intact vasopressin, with a half-life of 2.5 h. A loop formed by disulfide bond formation was regarded as one of the degradation-resistant factors. Hydrolysis of the peptides in this study took place through cleavage of various peptide bonds, and the strain B-9 may bear similarities to the neutral endopeptidase in terms of its broad selectivity.     

Synthesis of cyclic proline-containing peptides via ring-closing metathesis

[reaction: see text] Several dienes embedded in di- and tripeptides which incorporate proline have been prepared and subjected to ring-closing metathesis. Bicyclic peptides of well-defined amide geometry and of varying ring sizes were prepared. Several limitations of the cyclization step were revealed.     

Pictet-Spengler heterocyclizations via microwave-assisted degradation of DMSO

For the first time the ability of DMSO to decompose rapidly under microwaves was explored in order to perform heterocyclizations in good conditions. This modified Pictet-Spengler reaction allowed, in this case, the synthesis of rigidified melatonin analogues.     

Introduction of functional groups into peptides via N-alkylation

An optimized protocol for the mild and selective Fukuyama-Mitsunobu reaction was used for mono- and di- N-alkylation on solid support. Thereby, nonfunctionalized aliphatic and aromatic residues are quickly introduced into transiently protected, primary amines of a linear peptide. N-Alkylation can also be used to implement alkyl chains carrying (protected) functionalities suited for subsequent modification. Applicability of this method is demonstrated by various N-alkylated analogues of a cyclic CXCR4 receptor antagonist originally developed by Fujii et. al.