Simultaneous Analysis of Phosphorylated Peptides by MALDI-TOF-MS

Six peptides with various phosphorylation sensitivities for protein kinase A (PKA) were used for the simultaneous analysis of phosphorylated peptides using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry. The mixture of six peptides was reacted with PKA and was analyzed by MALDI-TOF mass spectrometry. The intensity of all peaks except one phosphorylated peptide peak was very low (<20%). Moreover, we examined whether the addition of diammonium citrate to CHCA matrix at concentrations of 1–20 mg mL^?1 can increase the peak intensity of peptides and phosphorylated peptides. The addition of diammonium citrate increased the peak intensity of peptides and phosphorylated peptides, but an increase in the intensity was unsatisfactory. Our study strongly suggests that MALDI-TOF mass spectrometry is not suitable for the simultaneous analysis of phosphorylated peptides.     

合成受体与模型磷酸化肽的相互作用机理

为了研究基于Zn2+-二甲基吡啶胺及胍羰基吡咯基团的配位型受体Zn Dpa G与磷酸化肽的相互作用机制,选取具有不同序列的磷酸化肽作用模型,采用等温滴定微量热法考察了Zn Dpa G与磷酸化肽的结合常数,研究了模型肽中磷酸基团的数量、密度及位置等因素对多肽与受体间结合强度的影响.结果表明,Zn Dpa G受体对双磷酸化肽结合能力显著高于单磷酸化肽,其结合常数可提高10~40倍,2个磷酸基团的距离越近,结合作用越强;而磷酸基团的位置显著影响受体与单磷酸化肽的结合强度.本研究结果为进一步优化磷酸化肽受体结构设计,实现肽与受体间高选择性识别提供了一定的理论依据.     

具有双活性序列的新型抗菌肽的设计及性质

设计合成了具有2个活性序列的线性和环状多肽及具有单个活性序列的短链多肽, 研究了它们的杀菌活性、 细胞毒性及溶血性. 结果表明, 线性肽和环状肽的杀菌活性高于短链肽. 利用计算模拟的方法计算了多肽与细菌细胞膜中一种重要的成分磷脂酰甘油(DMPG)的结合能. 结果表明, 多肽-DMPG的结合能与多肽的杀菌活性具有较高的相关性, 线性和环状多肽与DMPG的结合能大于短链肽. 线性和环状多肽均含有2个活性序列, 可提供多个荷正电氨基酸与荷负电的磷脂结合, 结合能较大, 杀菌活性较强. 采用模拟生物膜对其中几条多肽的作用机理进行了初步研究. 结果表明, 该类多肽有可能使正常哺乳动物细胞的细胞膜产生孔洞; 而对于细菌细胞膜, 多肽并未在膜上产生明显孔洞, 而是引起了细菌细胞膜的聚集.     

Reassessment of commercially available molecular weight standards for peptide sodium dodecyl sulfate-polyacrylamide gel electrophoresis using electroblotting and microsequencing

Myoglobin CNBr peptides, constituting the commercially available molecular weight calibration kits for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were analyzed by microsequencing after electroblotting on polyvinylidene difluoride (Immobilon) membranes. An obvious disagreement was found between peptide identification and the data provided by the manufacturers. We observed 6 peptides from Mr 2500 to 17,000 corresponding, in increasing size order, to the 3 peptides resulting from the total CNBr digestion, to 2 incompletely cleaved peptides and to the intact myoglobin. Using a corrected calibration curve, a linear relationship was established from Mr 6000 to 43,000 and a second one for shorter peptides. This method of electrophoresis and electroblotting, easily adapted for peptides, is a powerful tool for peptide identification correlated with size determination. It is especially useful for CNBr-cleaved peptides.     

Separation of peptides by HPLC using a surface-confined ionic liquid stationary phase

A butylimidazolium bromide surface-confined ionic liquid stationary phase was synthesized in-house. The synthesized phase was investigated for the separation of five peptides (Gly-Tyr, Val-Tyr-Val, leucine enkephalin, methionine enkephalin, and angiotensin-II). The peptides were successfully separated in less than 5 min. The effect of trifluoroacetic acid (TFA) on the separation of peptides was evaluated with results confirming that TFA was not acting as ion-pairing agent in separation of peptides on this phase.     

Fundamental studies on the electrokinetic transfer of net cationic peptides across supported liquid membranes

By the application of an electrical potential difference (25 V), 37 different peptides were extracted from 500 μL aqueous sample (10 mM formic acid, positive electrode), through a supported liquid membrane (SLM) impregnated in the walls of a porous hollow fiber, and into 25 μL aqueous acceptor solution (100 mM formic acid, negative electrode) present inside the lumen of the fiber. Most of the peptides were obtained by tryptic digestion of cytochrome c and bovine serum albumin, which yielded complex samples for extraction. Three different SLMs were utilized to correlate the peptides extractability with the highly variable physical-chemical properties of the peptides. The first SLM (pure eugenol) provided an electromembrane extraction system for hydrophobic and intermediate peptides (hydrophilicity values below 0.2), where the extraction of peptides into the SLM was mainly based on solvent interactions. The second SLM (1-octanol/di-isobutylketone/di-(2-ethylhexyl) phosphate) extracted both hydrophobic and hydrophilic peptides (hydrophilicity values in the range from -2 to+1) successfully, and the transfer of peptides was principally based on ionic interactions with di-(2-ethylhexyl) phosphate. The third SLM (1-octanol/15-crown-5 ether) was selective for hydrophobic peptides (negative hydrophilicity values), and complexation of the peptides with the crown ether was important for the migration of peptides into the acceptor solution.     

Multidimensional Design of Anticancer Peptides

The computer‐assisted design and optimization of peptides with selective cancer cell killing activity was achieved through merging the features of anticancer peptides, cell‐penetrating peptides, and tumor‐homing peptides. Machine‐learning classifiers identified candidate peptides that possess the predicted properties. Starting from a template amino acid sequence, peptide cytotoxicity against a range of cancer cell lines was systematically optimized while minimizing the effects on primary human endothelial cells. The computer‐generated sequences featured improved cancer‐cell penetration, induced cancer‐cell apoptosis, and were enabled a decrease in the cytotoxic concentration of co‐administered chemotherapeutic agents in vitro. This study demonstrates the potential of multidimensional machine‐learning methods for rapidly obtaining peptides with the desired cellular activities.     

Selection of possible marker peptides for the detection of major ruminant milk proteins in food by liquid chromatography-tandem mass spectrometry

The aim of this work was the determination of peptides, which can function as markers for identification of milk allergens in food samples. Emphasis was placed on two casein proteins (α- and β-casein) and two whey proteins (α-lactalbumin and β-lactoglobulin). In silico tryptic digestion provided preliminary information about the expected peptides. After tryptic digestion of four milk allergens, the analytical data obtained by combination of reversed-phase high performance liquid chromatography and quadrupole tandem mass spectrometry (LC-MS/MS) led to the identification of 26 peptides. Seven of these peptides were synthesized and used for calibration of the LC-MS/MS system. Species specificity of the selected peptides was sought by BLAST search. Among the selected peptides, only LIVTQTMK from β-lactoglobulin (m/z 467.6, charge 2+) was found to be cow milk specific and could function as a marker. Two other peptides, FFVAPFPEVFGK from α-casein (m/z 693.3, charge 2+) and GPFPIIV from β-casein (m/z 742.5, charge 1+), occur in water buffalo milk too. The other four peptides appear in the milk of other species also and can be used as markers for ruminant species milk. Using these seven peptides, a multianalyte MS-based method was developed. For the establishment of the method, it was applied at first to different dairy samples, and then to chocolate and blank samples, and the peptides could be determined down to 1 ng/mL in food samples. At the end, spiked samples were measured, where the target peptides could be detected with a high recovery (over 50%).     

Further investigation of a peptide extraction method with mesoporous silica using high‐performance liquid chromatography coupled with tandem mass spectrometry

Mobil Composition of Matter No. 41 (MCM‐41) was the most frequently used mesoporous silica material to extract peptides from complex biological samples. However, there were confusing extraction conditions and large extraction efficiency variance among related reports, which resulted from unclear understanding about the interaction between the material and peptides. In this study, the extraction mechanism was investigated with one set of tryptic peptides by using high‐performance liquid chromatography coupled with triple quadrupole mass spectrometry. Generally, hydrophobic interaction and electrostatic attraction were two major driving forces for extraction of peptides, while electrostatic repulsion greatly weakened the interaction between the material and peptides with isoelectric points below the pH. With most peptides positively charged and MCM‐41 slightly negatively charged, most efficient extraction was obtained at pH 3, and it was proved that electrostatic and hydrophobic interaction acted in synergy for extraction of all the peptides. A mixed solution of acetonitrile with buffers of high pH or ion strength was demonstrated to be favorable for elution, which performed much better than the commonly used eluate (mixture of acetonitrile with 0.1% trifluoroacetic acid). Finally, under optimum conditions, it was found that extraction efficiency of MCM‐41 for protein digest and human serum was greatly improved.     

The interaction of helical polypeptides with biological model membranes

Proton-decoupled solid-state ¹⁵N NMR spectroscopy was used to investigate helical peptides reconstituted into oriented phospholipid bilayers. Hydrophobic channel peptides such as the N-terminal region of Vpu of human immunodeficiency virus (HIV-1) adopt transmembrane orientations, whereas amphipathic peptide antibiotics are oriented parallel to the bilayer surface. The alignment of helical peptides in lipid membranes was analysed in some detail using model peptides. In particular, peptides with pH-dependent topology and a series of peptides that allow one to study the contributions of specific interactions were designed. The energies of transfer of several amino acids from the in-plane to transmembrane localisation were determined. In addition, the alignment of peptides and phospholipids under conditions of hydrophobic mismatch have been investigated in considerable detail.     

Easy amino acid sequencing of sulfonated peptides using post-source decay on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer equipped with a variable voltage reflector

Tryptic peptides were labeled with sulfonic acid groups at the N-termini using an improved chemistry. The derivatization was performed in common aqueous buffers on peptides adsorbed onto a ZipTip trade mark C(18), thus allowing simultaneous desalting/concentration of the sample. When only Arg-terminating peptides were considered, the procedure from adsorption onto the ZipTip until analysis by MALDI-PSD took about 10 min and several samples could be worked on in parallel. The resulting improved post-source decay (PSD) fragmentation produced spectra containing only y-ions. PSD amino acid sequencing of underivatized and derivatized synthetic peptides was compared. From the sequence information obtained from derivatized peptides isolated by ion selection from tryptic in-gel digests, a protein was correctly identified which was difficult to analyze from an unclear peptide mass fingerprint analysis. The method was also applied to the identification and localization of phosphorylated Ser and Tyr residues in native and synthetic peptides.     

赖氨酸胍基化对蛋白质组分析的影响

赖氨酸胍基化在蛋白质组学定性和定量研究中起着重要作用,本文系统分析了胍基化前后,HeLa细胞蛋白质经胰蛋白酶酶解产生的3种不同类型肽段的质谱鉴定情况,并探讨了不同肽段质谱响应改变的内在原因。发现赖氨酸在侧链能选择性地发生胍基化反应（其选择性达到96.8%）,转化为高精氨酸,碱性增强。因此在正离子质谱模式下,C端为赖氨酸的肽段产生了更多的y离子,提供了许多新的离子碎片信息。在鉴定结果中,此类肽段所占总肽段的比例由原来的51.7%上升为57.3%,并且有1015条新的肽段被检测到。对于不含有赖氨酸的肽段,其鉴定结果在胍基化前后基本没有变化。结果表明,胍基化可以在一定程度上提高质谱鉴定的灵敏度和互补性,提高蛋白质分析的覆盖率。     

Application of an anhydrotrypsin-immobilized precolumn for selective separation of peptides having arginine or lysine at their C-termini by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic (CS-HPLC) system which consisted of an anhydrotrypsin (AHT)-immobilized diol-silica precolumn and a reversed-phase analytical column was developed for the selective separation of peptides having Arg or Lys at their C-termini. Tuftsin (Thr-Lys-Pro-Arg) could be enriched almost quantitatively on the precolumn when loaded with water as a carrier solvent and the precolumn was washed with 10-30 mM acetate buffer (pH 5.0). An investigation of the affinity characteristics of 55 peptides to the AHT precolumn showed that among twelve peptides having Arg or ArgNH2 at their C-termini and more than four amino acid residues, ten were retained almost quantitatively on the precolumn, and eight out of nine peptides having Lys at their C-termini were less retained. The peptide having D-Arg at its C-termini was not retained. However, twelve out of thirty peptides having no Arg or Lys at their C-termini were also retained, but the retention was greatly decreased, in contrast to the Arg peptides, when the precolumn was washed with 20 mM calcium chloride solution. The results indicate that the CS-HPLC system equipped with an AHT precolumn offers new selectivity in the HPLC selectivity in the HPLC separation of peptides.     

Purification of Solid-Phase Synthesized Peptides on the Coil Planet Centrifuge

Abstract

The analytical flow-through coil planet centrifuge, an instrument for countercurrent chromatography, performs the preparative purification of synthetic peptides. Various two-phase solvent systems have been tried with either phase mobile to purify many synthesized peptides. A series of N-terminal fragment peptides of cholecystokinin octapeptide (CCK 26–33) were synthesized by solid-phase techniques and purified on the coil planet centrifuge. The peptides were sulfated and chromatographed again. For hydrophobic peptides, purification is effected in solvent systems with a mobile aqueous phase. The n-butanol, acetic acid and water system (4:1:5 by volume) with the lower phase mobile was utilized. For sulfated peptides, the neutral system, 0.2 M ammonium acetate and n-butanol was generally applied.     

Sortase A-catalyzed peptide cyclization for the synthesis of macrocyclic peptides and glycopeptides

A chemoenzymatic method was developed for the synthesis of macrocyclic peptides and glycopeptides. Sortase A was found to mediate either head to tail cyclization or oligomerization and then head to tail cyclization of peptides and glycopeptides, depending on the peptide length, to produce 15-mer or higher cyclic peptides and glycopeptides.     

Conductance of alpha-helical peptides trapped within molecular junctions

Self-assembled monolayers of alpha-helical peptides on a gold surface were employed as model systems for the investigation of mediated electron transfer. The peptides contained 14, 15, 16, and 17 amino acid residues. The measurements of electron transmission through single molecules of helical peptides were performed using scanning tunneling spectroscopy (STS). The molecules were trapped between the gold tip and the substrate. Electrical contact between the molecule and the gold probe was achieved by the use of peptides containing thiol groups present at each end of the helix. The conductance behavior of the peptides was examined as a function of tip-substrate distance at fixed bias voltage. Measurements performed with peptides containing different numbers of amino acid residues indicate that the distance dependence of electron transmission through an alpha-helix is weaker than that through simple n-alkyl bridges.     

An improved method for de novo sequencing of arginine-containing, Nalpha-tris(2,4,6-trimethoxyphenyl)phosphonium-acetylated peptides

An improved method for de novo sequencing of arginine-containing peptides modified with succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) is reported. A tagging reagent, TMPP-Ac-OSu, was introduced to improve the sequence analysis of peptides owing to the simplified fragmentation pattern. However, peptides containing arginine residues did not fragment efficiently even after TMPP-Ac modification at their N-termini. This report describes how fragmentation efficiency of TMPP-Ac-modified arginine-containing peptides was significantly improved by modifying the guanidino group on the side chain of arginine with acetylacetone.     

Simultaneous analysis by capillary electrophoresis of five amyloid peptides as potential biomarkers of Alzheimer's disease

We report here a CE method for the separation and quantitation of five amyloid peptides (Abeta1-42, 1-40, 1-39, 1-38, and 1-37) considered as potential biomarkers of Alzheimer's disease. These amyloid peptides have very similar structures. Sample preparation and storage conditions are critical parameters to ensure their solubility and to avoid the aggregation process in particular for Abeta1-42. Their solubility was found fully dependent on the NH(4)OH concentration that was employed initially to dissolve the lyophilized amyloid peptides. Conditions to achieve a full separation of these peptides were found using a dynamic coating with 1,4-diaminobutane (DAB). The linear decrease of their electrophoretic mobility highlighted an ion-pairing phenomenon between the peptides and DAB. The optimal background electrolyte was a 40 mM borate buffer, pH 9 containing 3 mM of DAB. Under these conditions, resolutions ranged from 1.3 to 2.4 with theoretical plates reaching 300,000. Under the retained conditions, we showed that adsorption of peptides to silica was negligible (recovery over 94.5%) and depletion effect of the background electrolyte was overcome. The method was finally validated in terms of linearity and repeatability and the limits of detection for the five Abeta peptides were estimated. The inter-day repeatability of the migration times was very satisfactory with RSDs less than 1.55%. The RSDs of the peak areas were below 5%. With this CE-UV method, limits of detection of the peptides ranged from 300 to 500 nM. We finally demonstrated that this method can be applied to real biological samples such as CSF.     

Single-molecule electrophoresis of beta-hairpin peptides by electrical recordings and Langevin dynamics simulations

We used single-channel electrical recordings and Langevin molecular dynamics simulations to explore the electrophoretic translocation of various beta-hairpin peptides across the staphylococcal alpha-hemolysin (alphaHL) protein pore at single-molecule resolution. The beta-hairpin peptides, which varied in their folding properties, corresponded to the C terminal residues of the B1 domain of protein G. The translocation time was strongly dependent on the electric force and was correlated with the folding features of the beta-hairpin peptides. Highly unfolded peptides entered the pore in an extended conformation, resulting in fast single-file translocation events. In contrast, the translocation of the folded beta-hairpin peptides occurred more slowly. In this case, the beta-hairpin peptides traversed the alphaHL pore in a misfolded or fully folded conformation. This study demonstrates that the interaction between a polypeptide and a beta-barrel protein pore is dependent on the folding features of the polypeptide.     

Effects of G-quartet DNA stationary phase destabilization on fibrinogen peptide resolution in capillary electrochromatography

The migration of fibrinogen peptides in capillaries coated with G-quartet-forming DNA oligonucleotides in open-tubular CEC (OTCEC) was studied, in order to investigate factors affecting the retention of peptides on G-quartet DNA stationary phases. At 25 degrees C, the peptides eluted in the same order in OTCEC using a two-plane G-quartet DNA stationary phase as in CZE, including two peptides that were completely overlapped. It was found that baseline resolution of the coeluting peptides could be achieved in the OTCEC experiment, but not in CZE, at run temperatures of 35-40 degrees C. A stationary phase formed by a scrambled-sequence oligonucleotide that does not form a G-quartet did not provide any resolution of the two coeluting peptides, even at the higher temperatures, indicating that some destabilization of the G-quartet enhances resolution but that some degree of G-quartet structure is necessary. The effects of destabilization were further explored through variation of the cations (sodium or potassium) used in attachment of the G-quartet oligonucleotide to the capillary surface and in the mobile-phase buffer. Resolution was lower when a more stable, four-plane G-quartet stationary phase was used, supporting the conclusion that some flexibility in the G-quartet structure facilitates differential interactions that resolve otherwise coeluting peptides. The increase in peptide resolution upon destabilization of the G-quartet structure could prove to be an important factor in the application of G-quartet DNA stationary phases for nonaffinity-based separation of native proteins and peptides.