Charge Derivatization Research Articles

Enhanced Electron Transfer Dissociation through Fixed Charge Derivatization of Cysteines

Electron transfer dissociation (ETD) has proven to be a promising new ion activation method for proteomics applications due to its ability to generate c- and z-type fragment ions in comparison to the y- and b-type ions produced upon the more conventional collisional activation of peptides. However, low precursor charge states hinder the success of electron-based activation methods due to competition from nondissociative charge reduction and incomplete sequence coverage. In the present report, the reduction and alkylation of disulfide bonds prior to ETD analysis is evaluated by comparison of three derivatization reagents: iodoacetamide (IAM), N,N-dimethyl-2-chloro-ethylamine (DML), and (3-acrylamidopropyl)-trimethyl ammonium chloride (APTA). While both the DML and APTA modifications lead to an increase in the charge states of peptides, the APTA-peptides provided the most significant improvement in percent fragmentation and sequence coverage for all peptides upon ETD, including formation of diagnostic ions that allow characterization of both the C- and N-termini. In addition, the formation of product ions in multiple charge states upon ETD is minimized for the APTA-modified peptides.

Tris(2,4,6-trimethoxyphenyl)methyl carbenium ion for charge derivatization of amines and amino acids

Primary and secondary alkyl amines substitute readily one of the para-methoxy substituents of tris(2,4,6-trimethoxyphenyl)methyl carbenium ion. With this rapid process, the preparation of permanently charged positive derivatives of amines, amino acids and small C-protected peptides is achieved. This is utilized here to facilitate the matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) analysis of these compounds.

The Combination of Electron Capture Dissociation and Fixed Charge Derivatization Increases Sequence Coverage for O-Glycosylated and O-Phosphorylated Peptides

Electron capture dissociation (ECD) has become an alternative method to collision-activated dissociation (CAD) to avoid gas-phase cleavage of post-translational modifications carried by side chains from the peptide backbone. Nonetheless, as illustrated herein by the study of O-glycosylated and O-phosphorylated peptides, the extent of ECD fragmentations may be insufficient to cover the entire peptide sequence and to localize accurately these modifications. The present work demonstrates that the derivatization of peptides at their N-terminus by a phosphonium group improves dramatically and systematically the sequence coverage deduced from the ECD spectrum for both O-glycosylated and O-phosphorylated peptides compared with their native counterparts. The exclusive presence of N-terminal fragments (c-type ions) in the ECD spectra of doubly charged molecular cations simplifies peptide sequence interpretation. Thus, the combination of ECD and fixed charge derivatization appears as an efficient analytical tool for the extensive sequencing of peptides bearing labile groups.

Improving de Novo Sequencing of Peptides Using a Charged Tag and C-Terminal Digestion

An improved method for peptide de novo sequencing by MALDI mass spectrometry is presented. The method couples a charge derivatization reaction with C-terminal digestion to modify tryptic peptides. The charge derivatization attaches a fixed charge group onto the N-termini of peptides, and the enzymatic digestion after the derivatization step removes C-terminal basic amino acid residues such as arginine and lysine. The fragmentation of the modified peptide(s) under low-energy CID conditions (MALDI Q-TOF mass spectrometer) yields a simplified yet complete ion series of the peptide sequence. The validity of the method is demonstrated by the results from several model protein digests, where peptide sequences were correctly deduced either manually or through an automated sequencing program.

Alternative Procedure for Charged Derivatization to Enhance Detection Responses of Steroids in Electrospray Ionization-MS

A derivatization procedure has been examined to enhance the electrospray ionization (ESI)-MS detectabilities of steroids that charged derivatization is not suitable for. The derivatization procedure with 2-hydrazinopyridine or isonicotinoyl azide was very effective for the sensitive detection of di-oxosteroids or di-hydroxysteroids, respectively, and the detection limits of the resulting derivatives were as low as about 2 fmol. The derivatives also provided intense characteristic product ions in the MS-MS, which are expected to be usable for the selected reaction monitoring mode.

Functional Proteome Analysis of the Banana Plant (Musa spp.) Using de Novo Sequence Analysis of Derivatized Peptides

We report the use of chemical derivatization with MALDI-MS/MS analysis for de novo sequence analysis. Using three frequently used homology-based search algorithms, we were able to identify more than 40 proteins from banana, a nonmodel plant with unsequenced genome. Furthermore, this approach allowed the identification of different isoforms. We also observed that the identification score obtained varied according to the position of the peptide sequences in the query using the MS-Blast algorithm.

Determination of salivary dehydroepiandrosterone using liquid chromatography–tandem mass spectrometry combined with charged derivatization

A sensitive liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method for the quantification of dehydroepiandrosterone (DHEA) in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with the permanently charged reagent, 2-hydrazino-1-methylpyridine (HMP), and subjected to LC–MS–MS. The derivatization with HMP was very effective for increasing the detectability of DHEA in the positive-ESI-MS. Quantification was based on the selected reaction monitoring and androsterone was used as an internal standard. This method allowed the reproducible and accurate quantification of the salivary DHEA using a 200-μl sample and the limit of quantitation for DHEA was 25 pg/ml. No significant matrix effect or change in the measured value by freeze/thaw repetition was observed. The developed method was applied to clinical studies, and produced satisfactory results.

Selective Identification and Quantitative Analysis of Methionine Containing Peptides by Charge Derivatization and Tandem Mass Spectrometry

To enable the development of a tandem mass spectrometry (MS/MS) based methodology for selective protein identification and differential quantitative analysis, a novel derivatization strategy is proposed, based on the formation of a "fixed-charge" sulfonium ion on the side-chain of a methionine amino acid residue contained within a protein or peptide of interest. The gas-phase fragmentation behavior of these side chain fixed charge sulfonium ion containing peptides is observed to result in exclusive loss of the derivatized side chain and the formation of a single characteristic product ion, independently of charge state or amino acid composition. Thus, fixed charge containing peptide ions may be selectively identified from complex mixtures, for example, by selective neutral loss scan mode MS/MS methods. Further structural interrogation of identified peptide ions may be achieved by subjecting the characteristic MS/MS product ion to multistage MS/MS (MS3) in a quadrupole ion trap mass spectrometer, or by energy resolved "pseudo" MS3 in a triple quadrupole mass spectrometer. The general principles underlying this fixed charge derivatization approach are demonstrated here by MS/MS, MS3 and "pseudo" MS3 analysis of side chain fixed-charge sulfonium ion derivatives of peptides containing methionine formed by reaction with phenacylbromide. Incorporation of "light" and "heavy" isotopically encoded labels into the fixed-charge derivatives facilitates the application of this method to the quantitative analysis of differential protein expression, via measurement of the relative abundances of the neutral loss product ions generated by dissociation of the light and heavy labeled peptide ions. This approach, termed "selective extraction of labeled entities by charge derivatization and tandem mass spectrometry" (SELECT), thereby offers the potential for significantly improved sensitivity and selectivity for the identification and quantitative analysis of peptides or proteins containing selected structural features, without requirement for extensive fractionation or otherwise enrichment from a complex mixture prior to analysis.

Determination of prostatic androgens in 10 mg of tissue using liquid chromatography–tandem mass spectrometry with charged derivatization

A practical liquid chromatography-tandem mass spectrometric (LC-MS-MS) method for the determination of prostatic 5alpha-dihydrotestosterone (DHT) and testosterone (T) has been developed. The prostatic androgens were extracted with MeOH-H2O (3:7, v/v), purified with an Oasis HLB cartridge, derivatized with the permanently charged reagent 2-hydrazino-1-methylpyridine (HMP), and subjected to LC-MS-MS analysis using electrospray ionization (ESI) operated in the positive ion mode. The derivatization with HMP was very effective at increasing the detectability using the positive-ESI-MS. The method allowed the reproducible and accurate quantification of ng g(-1) tissue levels of prostatic androgens in 10 mg of tissue. That is, the intra- and inter-assay coefficients of variation were below 8.1 and 9.3%, respectively, and the analytical recoveries of the androgens were quantitative. The limits of quantitation for DHT and T were both 1.0 ng g(-1) tissue. The developed method was used to determine DHT and T in the prostates of patients with benign prostatic hyperplasia and prostate cancer, and satisfactory results were obtained.

A new charge derivatization procedure for peptide sequencing

Derivatization of peptides by a trityl cation-containing group and collision-induced dissociation measurements of the cationized peptides results in informative spectra that simplify peptide sequencing.

Qualitative and quantitative analysis of small amine molecules by MALDI-TOF mass spectrometry through charge derivatization.

A pair of isotopically coded light and heavy reagents, tris(2,4,6-trimethoxyphenyl)phosphonium acetic acid N-hydroxysuccinimide esters (1 and 2) were synthesized and used to derivatize low molecular weight (<500 Da) molecules containing primary or secondary amine functional groups for MALDI-TOF MS analysis. The light and heavy reagents added a 573 and 600 Da positively charged tag to each analyte, respectively. In the presence of 10 times excess of tag reagents, the coupling reactions reached near completion within 10 min. The derivatization greatly facilitated MALDI analysis of small molecules and significantly improved the sensitivity of analysis, allowing a limit of detection in the low femtomole range. Additionally, the reaction mixtures were directly analyzed by MALDI without sample cleanup. The quantification of small molecules by MALDI-TOF MS was successfully achieved by analysis of isotopically coded light and heavy derivatives. MALDI-TOF quantitative analysis of a mixture of antibiotics yielded calibration curves in the concentration range from 0.3 to 30 pmol/microL with r2 values greater than 0.9995.

Charge derivatization by 4-sulfophenyl isothiocyanate enhances peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

High-sensitivity, rapid identification of proteins in proteomic studies normally uses a combination of one- or two-dimensional electrophoresis together with mass spectrometry. The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have increased its application in recent years. The most common method of 'peptide fingerprinting' often may not provide robust identification. Normally additional sequence information by post-source decay (PSD) MALDI-TOFMS provides additional constraints for database searches to achieve highly confident results. Here we describe a derivatization procedure to facilitate the acquisition of such sequence information. Peptide digests from a skin-expressed protein were modified with 4-sulfophenyl isothiocyanate. The resulting peptides carry a fixed negative charge at the N-terminal end and the resulting PSD spectrum is dominated by C-terminal y-type ions. The sequence information in most cases can be obtained manually or with simple programming tools. Methods of optimizing the procedure and increasing the sensitivity are discussed.

Investigation of the influence of charge derivatization on the fragmentation of multiply protonated peptides

The fragmentation of the multiply charged peptides b-chain of bovine insulin and glucagon have been investigated under low energy collision induced dissociation (CID) conditions using an electrospray ion trap mass spectrometer. The influence of charge state, specific amino acids such as aspartate or proline, the location of basic sites, and the derivatization on the fragmentation behavior has been the focus of interest. As a basis for understanding the fragmentation process, the concept of the mobile proton was applied. A set of different derivatives was used to manipulate the sites of protonation of the peptides in order to control and improve the fragmentation behavior. These results can be applied for de novo sequencing, although the sequence-specific fragmentation processes have significant influence on the dissociation behavior of the peptides.

Effect of charge derivatization in the determination of phosphorylation sites in peptides by electrospray ionization collision-activated dissociation tandem mass spectrometry

Effect of charge derivatization in the determination of phosphorylation sites in peptides by electrospray ionization collision-activated dissociation tandem mass spectrometry

Effect of charge derivatization in the determination of phosphorylation sites in peptides by electrospray ionization collision-activated dissociation tandem mass spectrometry

Effect of charge derivatization in the determination of phosphorylation sites in peptides by electrospray ionization collision-activated dissociation tandem mass spectrometry

Evaluation of charge derivatization of a proteolytic protein digest for improved mass spectrometric analysis: de novo sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry.

A simple mass spectrometric method to sequence a recombinant phosphoenolpyruvate carboxykinase of known structure and a novel variant of unknown structure isolated from Anaerobiospirillum succiniciproducens and Actinobacillus succinogenes 130Z, respectively, was evaluated. The proteolytic digests of the proteins were each chemically derivatized at the N-terminus by addition of a tris(trimethoxyphenyl)phosphoniumacetyl (TMPP(+)-Ac) group to produce peptides with a fixed positive charge. The derivatized digests were then partially separated by reversed-phase high-performance liquid chromatography. The fractions collected were subjected to matrix-assisted laser desorption/ionization post-source decay (MALDI/PSD) mass spectrometric analysis. The resulting spectra are sufficiently simple to allow the sequence to be read directly without extensive interpretation. This is in contrast to spectra of underivatized peptides obtained by MALDI/PSD or conventional tandem mass spectrometry, where full sequence interpretation can be challenging. Aided with a set of very simple established rules, it was shown that the sequence of TMPP(+)-Ac derivatives can be derived strictly from predictable fragment ion series. In most cases, this is sufficient to determine extensive, unambiguous, peptide sequences de novo. The partial sequence (35%) of the unknown phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes 130Z was obtained entirely by the mass spectrometric method evaluated here, which provided the basis for evaluating homology and for the design of oligonucleotide probes for cloning the corresponding gene.

Evaluation of charge derivatization of a proteolytic protein digest for improved mass spectrometric analysis: de novo sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry

Evaluation of charge derivatization of a proteolytic protein digest for improved mass spectrometric analysis: de novo sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry

Evaluation of charge derivatization of a proteolytic protein digest for improved mass spectrometric analysis: de novo sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry

Evaluation of charge derivatization of a proteolytic protein digest for improved mass spectrometric analysis: de novo sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry

Charge derivatization of peptides to simplify their sequencing with an ion trap mass spectrometer.

The low energy collision-induced dissociation of fixed-charge derivatives [tris(2,4,6-trimethoxyphenyl)phosphonium] of peptides was investigated using an electrospray ion trap mass spectrometer. The fixed charge directed the fragmentation pattern and generated solely N-terminal fragments with minimal internal rearrangement, regardless of the presence and position of basic amino acids in the peptide chain. Generally only b-type ions, accompanied by less intense a-type ions, were observed, depending on the collision energy. It was observed that the fixed charge controlled the fragmentation beyond typical MS/MS, and thus the capacity of the ion trap to perform multiple stage fragmentation (MS(n)) was found particularly useful for obtaining the complete sequence information of the peptides.

Charge derivatization of peptides for analysis by mass spectrometry

The analysis of peptide derivatives by fast atom bombardment, liquid secondary-ionization mass spectrometry, plasma desorption, electrospray ionization, and matrix-assisted laser desorption/ionization is reviewed. The fragmentation patterns of peptides and of charge-derivatized peptides are compared, and the proposed fragment ion structures are summarized. A variety of derivatization approaches and the distinguishing features of mass spectra produced from these derivatives are described. The most promising derivatization approaches are evaluated, and the strengths and limitations of these approaches are discussed. © 1999 John Wiley & Sons, Inc. Mass Spec Rev 17: 255–274, 1998