Analysis of cysteine-containing proteins using precolumn derivatization with <Emphasis Type="Italic">N</Emphasis>-(2-ferroceneethyl)maleimide and liquid chromatography/electrochemistry/mass spectrometry

N-(2-Ferroceneethyl)maleimide (FEM) is introduced as an electroactive derivatizing agent for thiol functionalities in proteins. Using appropriate reaction conditions, the derivatization is completed within five minutes and no unspecific labeling of free amino functions is observed. Liquid chromatography/electrochemistry/mass spectrometry was used to detect the reaction products. The reagent is a useful tool for determining the number of free thiol groups or the total number of free and disulfide-bound thiol groups in proteins. The electrochemical cell provides additional information, because the increase in mass spectrometric response upon electrochemical oxidation of the neutral ferrocene to the charged ferrocinium groups is monitored. The method was successfully applied to the analysis of native proteins and their tryptic digests. Dedicated to Prof. Werner Engewald on the occasion of his 70th birthday.     

Voltammetric Determination of Surface‐Confined Biomolecules with N‐(2‐Ethyl‐ferrocene)maleimide

A thiol‐specific electroactive cross‐linker, N‐(2‐ethyl‐ferrocene)maleimide (Fc‐Mi), has been used to tag surface‐confined peptides containing cysteine residues or oligodeoxynucleotides (ODNs) whose 3′ ends have been modified with thiol groups. The peptides studied herein include both the oxidized and reduced forms of glutathione and a hexapeptide. Cyclic voltammograms (CVs) of the Fc‐Mi groups attached to the surfaces were used to quantify the total number of cysteine residues that are tagged and/or can undergo facile electron transfer reactions with the underlying electrodes. A quartz crystal microbalance was used in conjunction with CV to estimate the total number of cysteine groups labeled by Fc‐Mi per peptide molecule. By comparing to mass spectrometric studies, it is confirmed that not all of the Fc‐Mi linked to the cysteine groups can participate in the electron transfer reactions. The methodology is further extended to the determination of ODN samples in a sandwich assay wherein the thiol linker on the 3′ end can be tagged with Fc‐Mi. The analytical performance was evaluated through determinations of a complementary ODN target and targets with varying numbers of mismatching bases. ODN samples as low as 10 fmol can be detected. Such a low detection level is remarkable considering that no signal amplification scheme is involved in the current method. The approach is shown to be sequence‐ and/or structure‐specific and does not require sophisticated instrumentation and complex experimental procedure.     

Design and synthesis of a solid-phase fluorescent mass tag

In this study, we demonstrate the design of a new solid-phase fluorescent mass tag (FMT) that contains the following features: (1) the FMT is synthesized using Fmoc chemistry which is simple, rapid, and cost-effective; (2) lysine is used as a uniformly labeled amino acid (using stable isotopes) to allow 8 Da difference between "heavy" and "light" tags; (3) a fluorescent molecule is coupled to the isotope tag that allows a tagged peptide to be detected by online fluorescence; and (4) an iodoacetyl reactive group provides cysteine reactivity. Using MALDI-TOF MS and HPLC, we show that the FMT reagent can be used to label standard cysteine-containing peptides as well as cysteine-containing peptides from a BSA tryptic digest.     

Design and synthesis of site directed maleimide bifunctional chelators for technetium and rhenium

A new family of heterobifunctional linkers (L1-L9) containing a terminus consisting of a tridentate donor set for coordination of the {M(CO)(3)}(+) core (M = Tc, Re), and a thiol reactive maleimide group has been prepared conveniently and in high yield under Mitsunobu reaction conditions by the coupling of an appropriate alcohol derivative with maleimide. The rhenium complexes [Re(CO)(3)(Lx)]Br (x= 1-9) were prepared in good yields from the reactions of the ligands and (NEt(4))(2)[Re(CO)(3)Br(3)] in refluxing methanol. The ligands and their Re complexes were characterized by (1)H and (13)C NMR, IR, and ESI-MS. Ligand L4 and [Re(CO)(3)(L5)]Br have been structurally characterized by X-ray crystallography. Photoexcitation of solutions of the complexes [Re(CO)(3)(Lx)]Br (x= 4-6) gives rise to intense and prolonged luminescence at room temperature (fluorescence lifetimes of ca. 16 micros). The ligands and their Re complexes react smoothly at the maleimide linker with sulfhydryl groups of peptides and proteins at room temperature in phosphate-buffered saline (PBS, pH 7.4) to form stable thioether bioconjugates. The photoluminescence properties of the labeled conjugates are similar to those of the parent complexes, but with even longer lifetimes. The ligands can also be labeled at room temperature with (99m)Tc to give chemically robust complexes. The corresponding hydrazinonicotinamide derivative N-[5-(6'-hydrazinopyridine-3'-carbonyl)aminopentyl]maleimide (L10) was also prepared. While coupling of L10 to cysteine ethylester and synthesis of the rhenium derivative [ReCl(3)(HYNIC-maleimide)(2)] were successfully accomplished, attempts to couple [ReCl(3)(HYNIC-maleimide)(2)] to glutathione or BSA yielded intractable mixtures.     

Facile chemoselective synthesis of dehydroalanine-containing peptides

Useful methodology is described for the synthesis of dehydroalanine residues (II) within peptides. The unnatural amino acid (Se)-phenylselenocysteine (I) can be incorporated into growing peptide chains via standard peptide synthesis procedures. Subsequent oxidative elimination affords a dehydroalanine at the desired position. The oxidation conditions are mild and tolerate functionalities commonly found in peptides, including variously protected cysteine residues. To illustrate its utility, cyclic lanthionines have been synthesized by this method.     

Mass spectrometric characterization of lipid-modified peptides for the analysis of acylated proteins

The analysis of acylated proteins by mass spectrometry (MS) has largely been overshadowed in proteomics by the analysis of glycosylated and phosphorylated proteins; however, lipid modifications on proteins are proving to be of increasing importance in biomedical research. In order to identify the marker ions and/or neutral loss fragments that are produced upon collision-induced dissociation, providing a means to identify the common lipid modifications on proteins, peptides containing an N-terminally myristoylated glycine, a palmitoylated cysteine and a farnesylated cysteine were chemically synthesized. Matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF-TOF), electrospray ionization quadrupole time-of-flight (ESI Q-TOF), and electrospray ionization hybrid triple-quadrupole/linear ion trap (ESI QqQ(LIT)) mass spectrometers were used for the analysis. The peptide containing the N-terminally myristoylated glycine, upon CID, produced the characteristic fragments a1 (240.4 Th) and b1 (268.4 Th) ions as well as a low-intensity neutral loss of 210 Da (C14H26O). The peptides containing a farnesylated cysteine residue fragmented to produce a marker ion at a m/z of 205 Th (C15H25) as well as other intense farnesyl fragment ions, and a neutral loss of 204 Da (C15H24). The peptides containing a palmitoylated cysteine moiety generated neutral losses of 238 Da (C16H30O) and 272 Da (C16H32OS); however, no marker ions were produced. The neutral losses were more prominent in the MALDI-TOF-TOF spectra, whereas the marker ions were more abundant in the ESI QqQ(LIT) and Q-TOF mass spectra.     

Diels-Alder ligation of peptides and proteins

The development of the Diels-Alder cycloaddition as a new method for the site-specific chemoselective ligation of peptides and proteins under mild conditions is reported. Peptides equipped with a 2,4-hexadienyl ester and an N-terminal maleimide react in aqueous media to give cycloadducts in high yields and depending on the amino acid sequence with high stereoselectivity. Except for the cysteine SH group the transformation is compatible with all amino acid side chain functional groups. For ligation to proteins the hexadienyl group was attached to avidin and streptavidin noncovalently by means of complex formation with a biotinylated peptide or by covalent attachment of a hexadienyl ester-containing label to lysine side chains incorporated into the proteins. Site-specific attachment of the hexadienyl unit into a Rab protein was achieved by means of expressed protein ligation followed by protection of the generated cysteine SH by means of Ellman's reagent. The protein reacted with different maleimido-modified peptides under mild conditions to give the fully functional cycloadducts in high yield. The results demonstrate that the Diels-Alder ligation offers an advantageous and technically straightforward new opportunity for the site-specific equipment of peptides and proteins with further functional groups and labels. It proceeds under very mild conditions and is compatible with most functional groups found in proteins. Its combination with other ligation methods, in particular expressed protein ligation is feasible.     

低浓度甲醛对多肽和蛋白化学修饰的质谱研究

采用基质辅助激光解析电离飞行时间质谱( MALDI-TOF MS)和纳升电喷雾四极杆飞行时间串联质谱( Nano-ESI -QTOF MS)技术,以标准肽段和流感病毒基质蛋白酶切肽段为模型,研究了甲醛对蛋白质和多肽主链的修饰作用。采用与实际病毒灭活过程一致的实验条件(4℃,0.025%(V/V)福尔马林(37%(w/w)甲醛溶液)处理72 h),进行甲醛与多肽的化学反应。结果表明,在实验条件下,甲醛能与标准肽段N端的氨基反应生成羟甲基加合物,再发生缩合反应生成亚胺,形成+12 Da的产物。此外,甲醛还能与标准肽段中的精氨酸、赖氨酸的侧链发生反应,生成+12 Da的反应产物。对流感病毒基质蛋白的酶切肽段与甲醛的反应的质谱分析结果显示,多数的肽段都生成了+24 Da的产物,质量的增加来源于肽段N端氨基(+12 Da)和C端精氨酸或赖氨酸的侧链(+12 Da)的贡献。此外,还观察到有一个漏切位点的肽段生成了+36 Da的产物。本研究结果表明,在实验条件下,低浓度甲醛主要与肽段和蛋白的N 端氨基,以及精氨酸和赖氨酸侧链发生反应。本研究为分析低浓度甲醛与蛋白质的反应产物提供了有效的质谱分析方法和解谱依据。     

Characterization of Peanut Protein Hydrolysate and Structural Identification of Umami-Enhancing Peptides

Umami peptides are naturally found in various foods and have been proven to be essential components contributing to food taste. Defatted peanut powder hydrolysate produced by a multiprotease (Flavorzyme, Alcalase, and Protamex) was found to elicit an umami taste and umami-enhancing effect. The taste profiles, hydrolysis efficiency, amino acids, molecular weight distribution, Fourier transform infrared spectroscopy (FT-IR), and separation fractions obtained by ultrafiltration were evaluated. The results showed that peanut protein was extensively hydrolyzed to give mainly (up to 96.84%) free amino acids and peptides with low molecular weights (<1000 Da). Furthermore, β-sheets were the major secondary structure. Fractions of 1–3000 Da and <1000 Da prominently contributed to the umami taste and umami enhancement. To obtain umami-enhancing peptides, these two fractions were further purified by gel filtration chromatography, followed by sensory evaluation. These peptides were identified as ADSYRLP, DPLKY, EAFRVL, EFHNR, and SDLYVR by ultra-performance liquid chromatography (UPLC), and had estimated thresholds of 0.107, 0.164, 0.134, 0.148, and 0.132 mmol/L, respectively. According to the results of this work, defatted peanut powder hydrolysate had an umami taste and umami-enhancing effect, and is a potential excellent umami peptide precursor material for the food industry.     

Controlling Peptide Self‐Assembly through a Native Chemical Ligation/Desulfurization Strategy

Self‐assembled peptides were synthesized by using a native chemical ligation (NCL)/desulfurization strategy that maintained the chemical diversity of the self‐assembled peptides. Herein, we employed oxo‐ester‐mediated NCL reactions to incorporate cysteine, a cysteine‐based dipeptide, and a sterically hindered unnatural amino acid (penicillamine) into peptides. Self‐assembly of the peptides resulted in the formation of self‐supporting gels. Microscopy analysis indicated the formation of helical nanofibers, which were responsible for the formation of gel matrices. The self‐assembly of the ligated peptides was governed by covalent and non‐covalent interactions, as confirmed by FTIR, CD, fluorescence spectroscopy, and MS (ESI) analyses. Peptide disassembly was induced by desulfurization reactions with tris(2‐carboxyethyl)phosphine (TCEP) and glutathione at 80 °C. Desulfurization reactions of the ligated peptides converted the Cys and penicillamine functionalities into Ala and Val moieties, respectively. The self‐supporting gels showed significant shear‐thinning and thixotropic properties.     

N-t-butyliodoacetamide and iodoacetanilide: two new cysteine alkylating reagents for relative quantitation of proteins

The synthesis and application of two new alkylating reagents, N-tert-butyl-2-iodoacetamide (N-t-butyliodoacetamide) and 2-iodo-N-phenylacetamide (iodoacetanilide), are described. N-t-Butyliodoacetamide and iodoacetanilide were synthesised to purity in their d(0)-light and in their respective d(9)- and d(5)-heavy forms. The newly synthesised reagents are covalently bound to peptides containing cysteines via an alkylation reaction. The mass differences of 5 and 9 Da avoid possible problems of overlapping isotope distribution. For each alkylated cysteine a peptide mass increases, respectively, by a multiple of 113 and 133 Da for the d(0)-light form of N-t-butyliodoacetamide and iodoacetanilide. These reagents can therefore replace common alkylating reagents in existing proteomics-based applications. Alkylated peptides increase in mass in the same mass range as amino acids and remain suitable for tandem mass spectrometry (MS/MS) data acquisition and analysis. The compounds are simple to use and derivatisation is based on widely applied alkylating procedures. Preliminary results show that these reagents can be applied for both protein quantitation and identification by peptide mass finger printing and/or MS/MS techniques. Using these chemicals and the suggested workflow enables the quantitative analysis of the whole protein sample and realises access to peptides that may contain potential post-translational modifications. Other approaches that incorporate a matrix-assisted laser desorption/ionisation (MALDI) interface prior to MS can take advantage of these chemicals, such as the molecular scanner.     

Arginine-mimic labeling with guanidinoethanethiol to increase mass sensitivity of lysine-terminated phosphopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) generally shows better mass sensitivity for arginine-terminated peptides than for lysine-terminated peptides, presumed to arise from the higher proton affinity of the guanidine group in arginine. Here, we report a new method for analyzing phosphopeptides in which phosphopeptides are labeled with a novel chemical tag, guanidinoethanethiol (GET), by a beta-elimination/Michael addition before MS analysis. GET labeling converts phosphoserine into guanidinoethylcysteine (Gec) containing a guanidine moiety, along with an increase in mass of 21.1 Da. GET-labeled peptides are detected by MALDI MS with greatly increased peak intensities compared to those of intact phosphopeptides. In particular, GET labeling of lysine-terminated phosphopeptides dramatically increased peak intensity. GET labeling of lysine-terminated phosphopeptides improved sensitivity up to 22 times compared to that of the corresponding aminoethanethiol (AET) labeling, in which AET was used as a labeling tag containing an amino group instead of the guanidine group. These results show the guanidine group plays a very important role in increasing the observed sensitivity of MALDI MS for labeled peptide, derivatized from serine-phosphorylated peptides.     

One-pot labeling and purification of peptides and proteins with fluorescein maleimide

Fluorescein labeling of peptides and proteins is required for numerous biophysical or biological experiments such as fluorescence microscopy, fluorescence resonance energy transfer (FRET) or fluorescence imaging. The commonly used strategy relied on the coupling of the dye reagent followed by a gel filtration to recover the labeled molecule. Here we report a simplified method for the labeling of peptides and proteins on a cysteine residue and their purification. The method is based on the precipitation of peptides and proteins in acetone, fluorescein maleimide being soluble in this solvent. The excess of dye is fully eliminated after a couple of acetone washes and the precipitated peptide or protein is readily recovered.     

Post-self-assembly covalent chemistry of discrete multicomponent metallosupramolecular hexagonal prisms

The multicomponent coordination-driven self-assembly of hexakis[4-(4-pyridyl)phenyl]benzene, cis-(PEt(3))(2)Pt(II)(OTf)(2), and amine- or maleimide-functionalized isophthalate forms discrete hexagonal prisms as single reaction products. The amino or maleimide groups decorating the isophthalate pillars of the prisms provide reactive sites for post-self-asssembly modifications. In this communication, we demonstrate that the hexagonal prisms can be functionalized without disrupting the prismatic cores, enabling the incorporation of new functionalities under mild conditions.     

Efficient identification and quantification of proteins using isotope-coded 1-(6-methylnicotinoyloxy)succinimides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We describe a convenient and useful method for the identification and relative quantification of proteins using light and heavy reagents, 1-(6-methylnicotinoyloxy)succinimides (6-CH(3)-Nic-NHS and 6-CD(3)-Nic-NHS, respectively). This method is based on the chemical derivatization of amino groups of tryptic peptides with these reagents, i.e., the basic moiety of the reagents thus incorporated into both the N-terminal amino group and the epsilon-amino group of the lysine residue would improve the ionization efficiency of tryptic peptides. An increase in protein sequence coverage is achieved by derivatization with these reagents or by combination of mass values before and after derivatization. Since a combination of 6-CH(3)-Nic-NHS and d(3)-labeled reagent (6-CD(3)-Nic-NHS) generates a 3 Da mass difference per reaction site, the d(3)-labeled reagent shifts the mass values of d(0)-labeled peptides according to the number of reactive amino groups in the peptides. In the case of tryptic peptides, the mass values of C-terminal arginine and lysine peptides are shifted by 3 and 6 Da, respectively. Further, the 3 Da mass difference between 6-CH(3)-Nic-NHS and 6-CD(3)-Nic-NHS offers a means for the relative quantification of protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.     

Improved sequencing of oxidized cysteine and methionine containing peptides using electron transfer dissociation

Oxidative modifications to the side chains of sulfur-containing amino acids often limit the number of product ions formed during collision-induced dissociation (CID) and thus make it difficult to obtain sequence information for oxidized peptides. In this work, we demonstrate that electron-transfer dissociation (ETD) can be used to improve the sequence information obtained from peptides with oxidized cysteine and methionine residues. In contrast to CID, ETD is found to be much less sensitive to the side-chain chemistry, enabling extensive sequence information to be obtained in cases where CID fails to provide this information. These results indicate that ETD is a valuable technique for studying oxidatively modified peptides and proteins. In addition, we report a unique and very abundant product ion that is formed in the CID spectra of peptides having N-terminal cysteine sulfinic acid residues. The mechanism for this unique dissociation pathway involves a six-membered cyclic intermediate and leads to the facile loss of NH(3) and SO(2), which corresponds to a mass loss of 81 Da. While the facile nature of this dissociation pathway limits the sequence information present in CID spectra of peptides with N-terminal cysteine sulfinic acid residues, extensive sequence information for these peptides can be obtained with ETD.     

Advantages of derivatization by osmium tetroxide and 2,2'-bipyridine for matrix-assisted laser desorption/ionization post-source decay fragment ion analysis of peptides

Matrix-assisted laser desorption/ionization--post-source decay (MALDI-PSD) fragment ion analysis is frequently used for peptide sequence determination. PSD fragmentation is often changed or improved in terms of, e.g., sequence coverage, after derivatization. In this work, the influence of modification by an osmium tetroxide-bipyridine reagent (Os,bipy) on the MALDI-PSD behaviour of peptides is studied. The reagent modifies peptides specifically at tryptophan residues and oxidizes methionine to methionine sulfone and cysteine to cysteic acid. As a result the masses of some of the fragments are specifically shifted in case of peptides containing a methionine by +32 Da and, in cases of peptides containing a cysteine residue, by +48 Da. In addition, due to the change in protonation properties of a peptide after oxidation, fragments containing cysteic acid are in most cases totally suppressed. This effect significantly facilitates peptide sequence determination. Improvement of MALDI-TOFMS and PSD analysis after the reaction with Os,bipy is demonstrated for examples involving derivatives of humanin, a novel neuroprotective peptide.     

LC-MS/MS Analysis and Comparison of Oxidative Damages on Peptides Induced by Pathogen Reduction Technologies for Platelets

Pathogen reduction technologies (PRT) are photochemical processes that use a combination of photosensitizers and UV-light to inactivate pathogens in platelet concentrates (PCs), a blood-derived product used to prevent hemorrhage. However, different studies have questioned the impact of PRT on platelet function and transfusion efficacy, and several proteomic analyses revealed possible oxidative damages to proteins. The present work focused on the oxidative damages produced by the two main PRT on peptides. Model peptides containing residues prone to oxidation (tyrosine, histidine, tryptophane, and cysteine) were irradiated with a combination of amotosalen/UVA (Intercept process) or riboflavin/UVB (Mirasol-like process). Modifications were identified and quantified by liquid chromatography coupled to tandem mass spectrometry. Cysteine-containing peptides formed disulfide bridges (R-SS-R, ?2 Da; favored following amotosalen/UVA), sulfenic and sulfonic acids (R-SOH, +16 Da, R-SO₃H, +48 Da, favored following riboflavin/UVB) upon treatment and the other amino acids exhibited different oxidations revealed by mass shifts from +4 to +34 Da involving different mechanisms; no photoadducts were detected. These amino acids were not equally affected by the PRT and the combination riboflavin/UVB generated more oxidation than amotosalen/UVA. This work identifies the different types and sites of peptide oxidations under the photochemical treatments and demonstrates that the two PRT may behave differently. The potential impact on proteins and platelet functions may thus be PRT-dependent.

Quantitative proteomics by fluorescent labeling of cysteine residues using a set of two cyanine‐based or three rhodamine‐based dyes

Despite all remarkable progress in gel‐based proteomics in recent years, there is still need to further improve quantification by decreasing the detection limits and increasing the dynamic range. These criteria are achieved best by fluorescent dyes that specifically stain the proteins either by adsorption after gel electrophoresis (in‐gel staining) or covalent coupling prior to gel electrophoresis (in‐solution staining). Here we report a multiplex analysis of protein samples using maleimide‐activated cyanine‐based (Cy3 and Cy5) and rhodamine‐based dyes (Dy505, Dy535, and Dy635) to permanently label all thiol‐groups of cysteine‐containing proteins. The detection limits in SDS‐PAGE were about 10 ng per band and even 2 ng for BSA due to its high content of cysteine residues. Thus only 5 μg protein of a mouse brain homogenate were analyzed by 2‐DE. Both cyanine‐ and rhodamine‐based dyes also stained proteins that did not contain cysteines, probably by reaction with amino groups. This side reactivity did not limit the method and might even extend its general use to proteins missing cysteine residues, but at a lower sensitivity. The dynamic range was more than two orders of magnitude in SDS‐PAGE and the Dy‐fluorophores did not alter the mobility of the tested proteins. Thus, a mixture of Dy505‐, Dy555‐, and Dy635‐labeled Escherichia coli lysates were separated by 2‐DE in a single gel and the three spot patterns relatively quantified.     

Normalization of relative peptide ratios derived from in-gel digests: applications to protein variant analysis at the peptide level

The ability to detect protein variants and post-translational modifications by mass spectrometry has become increasingly important. Unfortunately, the ability to detect variants in large intact proteins (>80,000 Da) is limited. Even in the analysis of smaller proteins, algorithms are required to determine the presence of a 2 Da mass shift in an intact 13 kDa protein because the isotopic distribution of the multiply charged ions of the variant overlaps the wild-type distribution. Fortunately, most modern instruments are capable of detecting variants in tryptic peptides derived from intact proteins. If a single common variant protein is known, the presence of a variant tryptic peptide can be easily demonstrated. A more difficult issue is the case where a multiplicity of peptides with multiple amino acid substitutions can be associated with pathology. In these cases a decrease in the relative amount of a variant peptide relative to other internal tryptic fragments would be diagnostic. However, the variability associated with the analysis of in-gel or solution digests of proteins, related to efficiencies in digestion, extraction and ionization, confounds variant analysis at the peptide level. A strategy was developed to normalize for this variability by utilizing multiple isotopically labeled internal standards for multiple peptides derived from the same protein. Erythrocyte spectrin from 36 normal and 25 abnormal osmotic fragility samples was analyzed as a test case. Three isotopically labeled target peptides comprising the alpha/beta-spectrin self-association sites were added to purified digested alpha-spectrin. The utilization of multiple internal standards demonstrates the capability to normalize for sample variability due to ionization efficiency, solvent effects, digestion and extraction efficiency.