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。     

In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics

We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100 mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the epsilon-amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b-ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9- to D9-trimethyl-labeled peptides of beta-casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes.     

酶切过程中肽段过烷基化对蛋白质定性和定量分析的影响

蛋白质的还原-烷基化是蛋白质酶切中的重要步骤,常用的烷基化试剂是碘乙酰胺(IAA),但是IAA除了和半胱氨酸发生反应,也可能和其他多种氨基酸发生副反应。我们模拟常规的酶切条件,系统地研究了蛋白质真实酶切时所有酶切肽段发生烷基化的情况。结果表明,多种氨基酸可以发生烷基化,其趋势为:半胱氨酸>肽段N端氨基酸>天冬氨酸>谷氨酸>组氨酸>天冬酰胺>赖氨酸>酪氨酸,同时也发现同一肽段上的氨基酸烷基化具有排他性和聚集性。根据定性结果,采用质谱多反应监测(MRM)技术对多个肽段进行了定量分析,评估了过烷基化对蛋白质定量分析的影响。该研究结果表明,过量的烷基化修饰对蛋白质的定性与定量分析都可能产生较大影响。在蛋白质组学研究的样本处理流程中,应避免样本的过烷基化。     

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.     

Lysine peroxycarbamates: free radical-promoted peptide cleavage

Strategies are reported that combine in one step a predictable chemical-based protein digestion with mass spectrometry. Lysine residue amino groups in peptides and proteins are modified by reaction with a peroxycarbonate derived from p-nitrophenol, and tert-butyl hydroperoxide. The peroxycarbonate reacts with lysine residues in peptides and proteins, and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID). Observed fragmentation of the peptides involves apparent free radical processes including Hofmann-L?ffler-type rearrangements that lead to peptide chain fragmentation. Strategies for directed cleavage of peptides by free radical promoted processes are feasible, and such strategies may well simplify schemes for protein analysis.     

Modification of N-terminal α-amino groups of peptides and proteins using ketenes

A method of highly selective N-terminal modification of proteins as well as peptides by an isolated ketene was developed. Modification of a library of unprotected peptides XSKFR (X varies over 20 natural amino acids) by an alkyne-functionalized ketene (1) at room temperature at pH 6.3 resulted in excellent N-terminal selectivity (modified α-amino group/modified ε-amino group = >99:1) for 13 out of the 20 peptides and moderate-to-high N-terminal selectivity (4:1 to 48:1) for 6 of the 7 remaining peptides. Using an alkyne-functionalized N-hydroxysuccinimide (NHS) ester (2) instead of 1, the modification of peptides XSKFR gave internal lysine-modified peptides for 5 out of the 20 peptides and moderate-to-low N-terminal selectivity (5:1 to 1:4) for 13 out of the 20 peptides. Proteins including insulin, lysozyme, RNaseA, and a therapeutic protein BCArg were selectively N-terminally modified at room temperature using ketene 1, in contrast to the formation of significant or major amounts of di-, tri-, or tetra-modified proteins in the modification by NHS ester 2. The 1-modified proteins were further functionalized by a dansyl azide compound through click chemistry without the need for prior treatment.     

Fractionation-free negative enriching for in-depth C-terminome analysis

Herein, we developed a fractionation-free negative enriching method incorporating methylamidation, siteselective dimethylation and aldehyde resin coupling(MADMAR) for in-depth C-terminome analysis. The methylamidation blocked the free carboxyl group on proteins first, followed by Lys C digestion of methylamidated proteins. Then, the site-selective dimethylation blocked the N-terminal amino group of the digested peptides without affecting the amino groups of lysine. Finally, the aldehyde resin wa...     

A novel method for identification and relative quantification of N-terminal peptides using metal-element-chelated tags coupled with mass spectrometry

By means of the reaction between a DOTA-NHS-ester bifunctional reagent and N-terminal peptides of proteins,and then chelation of lanthanide metal ions as tags,we established a novel method for the identification of N-terminal peptides of proteins and their relative quantification using metal-element-chelated tags coupled with mass spectrometry.The experimental results indicate that metal elements are able to completely label N-terminal peptides at the protein level.The N-terminal peptides are enriched as the peptides digested with trypsin are selectively eliminated by isothiocyanate-coupled silica beads.We successfully identified the N-terminal peptides of 158 proteins of Thermoanaerobacter tengcongensis incubated at 55 and 75°C,among which N-terminal peptides of 24 proteins are partially acetylated.Moreover,metal-element tags with high molecule weights make it convenient for N-terminal peptides consisting of less than 6 amino acids to be identified;these make up 55percent of the identified proteins.Finally,we developed a general approach for the relative quantification of proteins based on N-terminal peptides.We adopted lysozyme and ribonuclease B as model proteins;the correlation coefficients(R2)of the standard curves for the quantitative method were 0.9994 and 0.9997,respectively,with each concentration ratio ranging from0.1 to 10 and both relative standard derivations(RSD)measured at less than 5%.In T.tengcongensis at two incubation temperatures,80 proteins possess quantitative information.In addition,compared with the proteins of T.tengcongensis incubated at 55°C,in T.tengcongensis incubated at 75°C,7 proteins upregulate whereas 16 proteins downregulate,and most differential proteins are related to protein synthesis.     

Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry

Guanidination of the epsilon-amino group of lysine-terminated tryptic peptides can be accomplished selectively in one step with O-methylisourea hydrogen sulfate. This reaction converts lysine residues into more basic homoarginine residues. It also protects the epsilon-amino groups against unwanted reaction with sulfonation reagents, which can then be used to selectively modify the N-termini of tryptic peptides. The combined reactions convert lysine-terminated tryptic peptides into modified peptides that are suitable for de novo sequencing by postsource decay matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The guanidination reaction is very pH dependent. Product yields and reaction kinetics were studied in aqueous solution using either NaOH or diisopropylethylamine as the base. Methods are reported for derivatizing and sequencing lysine-terminated tryptic peptides at low pmole levels. The postsource decay (PSD) MALDI tandem mass spectra of a model peptide (VGGYGYGAK), the homoarginine analog and the sulfonated homoarginine analog are compared. These spectra show the influence that each chemical modification has on the peptide fragmentation pattern. Finally, we demonstrate that definitive protein identifications can be achieved by PSD MALDI sequencing of derivatized peptides obtained from solution digests of model proteins and from in-gel digests of 2D-gel separated proteins.     

Changes in Protein Composition in Erythrocyte Membrane of Ethanol—Poisoned Rats After Administration of Teas

Selective hydrolysis of the protein to peptides, their resolution by chromatography and determination of the amino-acid sequence in individual peptides is one method applied to studies of their modifications and mutations in pathological conditions. The purpose of this work is the determination of the green and black tea effect on peptides and their amino acid contents in integral erythrocyte membrane proteins in ethanol intoxicated rats. It was shown that ethanol intoxication causes a decrease in peptides and their amino acid contents in comparison with the control group. The ingestion of green (black) tea with ethanol partially prevents these ethanol-induced changes.     

Liquid chromatography with complementary electrospray and inductively coupled plasma mass spectrometric detection of ferrocene-labelled peptides and proteins

Succinimidylferrocenyl propionate (SFP) is introduced as labelling agent for amino functions in peptides and proteins. The resulting derivatives are characterised by considerably lower polarity compared with the native analytes and can thus be well separated by means of reversed phase liquid chromatography (RP-LC). The reaction products are characterised by electrospray ionisation mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). A further advantage of the method is a simple and straightforward derivatisation protocol. Different basic and acidic model proteins as lysozyme, ß-lactoglobulin A and insulin were derivatised using SFP. Furthermore, the first dual-labelling strategy of thiol and amino groups with ferrocene-based reagents is presented. Whereas the amino groups were derivatised with SFP, the thiol groups were functionalised by reaction with ferrocenecarboxylic acid(2-maleimidoyl)ethylamide. Again, LC/ESI-MS is a suitable tool to characterise the modified peptides and proteins.     

PHOTOCHEMICAL ADDITION OF AMINO ACIDS AND PEPTIDES TO DNA

Abstract— The quantum yields for photochemical addition of twenty of the amino acids commonly occurring in proteins to denatured calf thymus DNA have been determined in deoxygenated phosphate buffer at λ 254 nM and pH 7 using a fluorescamine assay technique. Fifteen were found to be reactive, with cysteine, lysine, phenylalanine, tryptophan and tyrosine being the most reactive. Alanine, aspartic acid, glutamic acid, serine and threonine were unreactive. Analogous quantum yields for a series of eighteen peptides of the form glycyl X (X being one of the commonly occurring amino acids) were also determined, along with the corresponding quantum yields for L-alanyl-L-alanine, L-alanyl-L-tryptophan, L-seryl-L-seryl-L-serine, L-threonyl-L-threonyl-L-threonine, and L-cystine- bis -glycine. All of the peptides were found to be reactive. The modified amino acids N^ε-methyllysine, N^ε, N^ε, N^ε-trimethyllysine and N^ε-acetyllysine, all occurring in minor amounts in the histone group of chromosomal proteins, were also found to be reactive as was N^α-acetyllysine. The quantum yields for photoaddition of a selected group of amino acids and peptides to denatured DNA and native DNA are compared. In some cases higher quantum yields for photoaddition to denatured DNA are observed while in other cases the reverse is true. The effect of oxygen on the quantum yields for photoaddition of selected peptides to DNA was examined. While for most systems studied the amount of reaction in aerated systems was less than in deoxygenated systems, in the case of glycyl-L-phenylalanine the reverse was true.     

A method for rapidly confirming protein N-terminal sequences by matrix-assisted laser desorption/ionization mass spectrometry

The N-terminal sequence is important for the identification of a protein and the confirmation of its N-terminal processing. Although mass spectrometry (MS) is a sensitive and high-throughput method to sequence and identify peptides and proteins, N-terminal peptides, diluted among most of the peptides that do not originate at the N-termini, are not easy to identify directly with MS. To develop a simple and rapid method to identify and sequence the N-terminal peptide of a protein, a new strategy based on specific sulfonation of terminal amino groups and selective monitoring of the sulfonated peptide was introduced. After a protein had been guanidinated, 2-sulfobenzoylated, and reduced, it was digested with trypsin and analyzed by MS. Because of the strong acidity of sulfonic groups and the specific sulfonation of alpha-amino groups, the sulfonated N-terminal peptide dominated as base peak in the negative mode peptide mass fingerprint (PMF) and was easy to identify. The N-terminal peptide was then selected as precursor ion for tandem mass spectrometric (MS/MS) analysis. Four proteins were tested with this method and their N-terminal peptides were successfully recognized and sequenced. The results suggest that the addition of a sulfonic acid group facilitates the identification and de novo sequencing of N-terminal peptides.     

Biotinylation of Peptides/Proteins Using Biocytin Hydrazide

Biocytin hydrazide is widely used to biotinylate the carbohydrate moieties of glycoproteins. In this study, however, biocytin hydrazide was found to be able to directly biotinylate peptides and proteins. This phenomenon may cause false identification of non‐glycopeptides/non‐glycoproteins as glycopeptides/glycoproteins. Here, we report a systematic investigation of the reaction of peptides/proteins with biocytin hydrazide. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry is used to analyze the biotinylation reaction between peptides/proteins and biocytin hydrazide. Peptides/proteins were reacted with biocytin hydrazide in diverse solvent systems with different biocytin hydrazide concentrations for up to 96 h at temperatures ranging from 4 °C to 65 °C. Singly biotinylated or multiply biotinylated peptides/proteins are observed. The efficiency of the biotinylation reaction increases with higher temperature, higher biocytin hydrazide concentration, or longer reaction time. The influence of buffer pH on the biotinylation reaction of peptides/proteins is less pronounced. The biotinylation efficiency is optimum at neutral pH. Data suggests that the peptides are biotinylated as efficiently as proteins. The observation that peptides/proteins condense only with biocytin hydrazide, 2‐iminobiotin hydrazide, adipic dihydrazide and phenyl hydrazine but not with biocytin HCl and 2‐iminobiotin, indicates that the biotinylation reaction of peptides/proteins occurs with the hydrazide moiety but not with biotin moiety of the biotinylated reagent. The postsource decay data of biotinylated P₁₄R indicates that biocytin hydrazide condenses with the guanidino group of arginine's side chain of P₁₄R, indicating that besides N‐terminal and lysine residue of peptides/proteins, arginine residue is capable of reacting with biocytin hydrazide.     

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.     

Enzymatic Preparation of Bioactive Peptides Exhibiting ACE Inhibitory Activity from Soybean and Velvet Bean: A Systematic Review

The Angiotensin-I-converting enzyme (ACE) is a peptidase with a significant role in the regulation of blood pressure. Within this work, a systematic review on the enzymatic preparation of Angiotensin-I-Converting Enzyme inhibitory (ACEi) peptides is presented. The systematic review is conducted by following PRISMA guidelines. Soybeans and velvet beans are known to have high protein contents that make them suitable as sources of parent proteins for the production of ACEi peptides. Endopeptidase is commonly used in the preparation of soybean-based ACEi peptides, whereas for velvet bean, a combination of both endo- and exopeptidase is frequently used. Soybean glycinin is the preferred substrate for the preparation of ACEi peptides. It contains proline as one of its major amino acids, which exhibits a potent significance in inhibiting ACE. The best enzymatic treatments for producing ACEi peptides from soybean are as follows: proteolytic activity by Protease P (Amano-P from Aspergillus sp.), a temperature of 37 °C, a reaction time of 18 h, pH 8.2, and an E/S ratio of 2%. On the other hand, the best enzymatic conditions for producing peptide hydrolysates with high ACEi activity are through sequential hydrolytic activity by the combination of pepsin-pancreatic, an E/S ratio for each enzyme is 10%, the temperature and reaction time for each proteolysis are 37 °C and 0.74 h, respectively, pH for pepsin is 2.0, whereas for pancreatin it is 7.0. As an underutilized pulse, the studies on the enzymatic hydrolysis of velvet bean proteins in producing ACEi peptides are limited. Conclusively, the activity of soybean-based ACEi peptides is found to depend on their molecular sizes, the amino acid residues, and positions. Hydrophobic amino acids with nonpolar side chains, positively charged, branched, and cyclic or aromatic residues are generally preferred for ACEi peptides.     

Oxidative modification of native protein residues using cerium(IV) ammonium nitrate

A new protein modification strategy has been developed that is based on an oxidative coupling reaction that targets electron-rich amino acids. This strategy relies on cerium(IV) ammonium nitrate (CAN) as an oxidation reagent and results in the coupling of tyrosine and tryptophan residues to phenylene diamine and anisidine derivatives. The methodology was first identified and characterized on peptides and small molecules, and was subsequently adapted for protein modification by determining appropriate buffer conditions. Using the optimized procedure, native and introduced solvent-accessible residues on proteins were selectively modified with polyethylene glycol (PEG) and small peptides. This unprecedented bioconjugation strategy targets these under-utilized amino acids with excellent chemoselectivity and affords good-to-high yields using low concentrations of the oxidant and coupling partners, short reaction times, and mild conditions.     

Catalysis of Hydrogen Evolution by Polylysine,Polyarginine and Polyhistidine at Mercury Electrodes

Protein catalyzed hydrogen evolution reaction at mercury‐containing electrodes controlled by constant‐current chronopotentiometric stripping (CPS) is representing a new tool useful in protein research. The resulting CPS peak H is sensitive to changes in the protein structure and its amino acid composition. Besides CPS, cyclic voltammetry appears to be useful for study of poly(amino acids) as an intermediate model system between peptides and macromolecular proteins. Here we show that similarly as arginine in polyarginine and lysine in polylysine also histidine residues in polyhistidine contribute to the catalysis of hydrogen evolution under the given conditions. Peak potentials of individual poly(amino acids) are different and depend on the type of amino acid residues.     

Sortase-mediated protein ligation: a new method for protein engineering

Sortase (SrtA), a transpeptidase from Staphylococcus aureus, catalyzes a cell-wall sorting reaction at an LPXTG motif by cleaving between threonine and glycine and subsequently joining the carboxyl group of threonine to an amino group of pentaglycine on the cell wall peptidoglycan. We have applied this transpeptidyl activity of sortase to in vitro protein ligation. We found that in the presence of sortase, protein/peptide with an LPXTG motif can be specifically ligated to an aminoglycine protein/peptide via an amide bond. Additionally, sortase can even conjugate substrates such as (d)-peptides, synthetic branched peptides, and aminoglycine-derivatized small molecules to the C terminus of a recombinant protein. The sortase-mediate protein ligation is robust, specific, and easy to perform, and can be widely applied to specific protein conjugation with polypeptides or molecules of unique biochemical and biophysical properties.     

Effects of an arginine-selective tagging procedure on the fragmentation behavior of peptides studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS)

The presence of arginine as the naturally occurring amino acid with the highest gas-phase basicity strongly influences the fragmentation behavior of peptides undergoing collision-induced dissociation. Using a derivatization procedure recently developed in our group, based on a reversible reaction of the guanidino group with 2,3-butanedione and an arylboronic acid, we examined how this label affects the fragmentation patterns of labeled versus unlabeled peptides in MS/MS experiments. As part of this fundamental study, two groups of model peptides (angiotensins and bradykinins) as well as tryptic peptides were labeled according to our protocol and subjected to collision-induced dissociation (CID) in both a triple quadrupole and a quadrupole ion trap instrument. It was found that for angiotensins containing an AspArg sequence, C-terminal cleavage at Asp that occurs for native peptides was completely inhibited in Arg-labeled peptides. For bradykinins and peptides obtained from tryptic digests of standard proteins, some sample peptides were little affected by the tagging of arginine residues. Others, in contrast, exhibited an almost total loss of nonspecific backbone cleavage and their fragment ion spectra were dominated by loss of the arginine tag. These and other experimental results are discussed in view of the nature of the arginine tag and the concept of proton mobility.