Identification of formylglycine in sulfatases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

C(alpha)-Formylglycine, the catalytic amino acid residue in the active site of sulfatases, is generated by post-translational modification of a cysteine or serine residue. We describe a highly sensitive procedure for the detection of C(alpha)-formylglycine-containing peptides in tryptic digests of sulfatase proteins. The protocol is based on the formation of hydrazone derivatives of C(alpha)-formylglycine-containing peptides when using dinitrophenylhydrazine as a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives desorb and ionize with high efficiency and can be detected in the sub-femtomole range. The presence of C(alpha)-formylglycine is indicated by a mass increment of 180.13 u, corresponding to the hydrazone moiety, and also by a unique C-terminal fragment ion, characteristic of sulfatases, that becomes prominent in MALDI post-source decay mass spectra of the hydrazone derivatives.     

Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry

A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.     

Dehydrogenation of tertiary amines in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis of various compounds synthesized in our laboratory, strong [M - H]+ ion peaks were often observed for the molecules with tertiary amino groups. In this work, the MALDI TOF MS behavior of two groups of compounds that incorporate tertiary amino moieties was investigated. One group is bisurea dimethylanilines (BUDMAs) prepared for the study of molecular recognition in thermoplastic elastomers, and the other group is the poly(propylene imine) diaminobutane dendrimers. The results clearly demonstrate the appearance of the [M - H]+ ions. In order to understand the possible mechanisms for the generation of these ions, a series of model compounds, ranging from primary to tertiary amines, were investigated. Unlike the tertiary amines, no [M - H]+ ion peaks were recorded for the primary amines, and only barely detectable ones, if any, for some secondary amines. It appears that the tertiary amino groups play an important role in the formation of these ions. In addition to MALDI TOF MS analysis, these samples were also applied to electrospray ionization (ESI) MS where no [M - H]+ ions were observed. The results indicate that the generation of [M - H]+ ion is due to the unique MALDI conditions and is likely to be formed via dehydrogenation of a protonated tertiary amine resulting in an N=C double bond. The absence of [M - H]+ ion peaks for the primary and secondary amines is probably because upon their formation these ions could easily transfer one proton to the corresponding amines in the MALDI gas-phase plume, yielding neutral imines that cannot be detected by MS.     

Detection of water-soluble vitamins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using porphyrin matrices

The detection of water-soluble vitamins B(1), B(2), B(6), B(12) and C by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) was attempted by studying 17 porphyrin matrices. Comparative studies of porphyrin matrices, useful mass spectral window, matrix/analyte molar ratio (M/A), ultraviolet-visible absorption characteristics and quantitative results were made. Most porphyrin matrices provide a useful mass spectral window in the low-mass range. The optimal M/A increases with increasing molecular mass of the vitamin. Vitamin B(12) possesses the highest molecular mass and requires a higher M/A. The presence of hydroxyl or carboxyl groups in the porphyrin is an indicator of a useful MALDI matrix. Vitamins B(2) and B(6) were readily ionized upon irradiation with a 337 nm laser without the use of any porphyrin matrix. Improved linearity and sensitivity of the calibration curves were obtained with samples prepared with a constant M/A. The limits of detection and quantitation are at the picomole level. The results indicate that MALDI-TOFMS with porphyrin matrices is a rapid and viable technique for the detection of low molecular mass water-soluble vitamins.     

Characterization of some synthetic Ru and Ir complexes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied to the analysis of Ru(OCOCF(3))(2)(CO)(PPh(3))(2), Ru(OCOC(3)F(7))(2)(CO)(PPh(3))(2), Ir(tBuppy)(3) and Ir(ppy)(2)(acac) complexes. A troublesome problem in the MALDI-TOFMS characterization of these metal complexes is the possible replacement of complex ligands by matrix. In this contribution, 10 matrices, ranging from acidic to basic, were investigated: alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), dithranol, 2,4,6-trihydroxyactophenone (THAP), 6-azo-2-thiothymine (ATT), norharman, 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB), 4-nitroaniline (NA) and 2-amino-5-nitrophyridine (ANP). With most of the matrices, including the neutral and basic ones, matrix substitution of ligand could clearly be detected. Based on the experimental results, possible mechanisms of matrix substitution were discussed. It was demonstrated that the ligand exchange process might also occur through the gas-phase reactions initiated by laser shots. Among the matrices tested, DCTB was found to be the best one for the complexes that are prone to ligand exchange by matrix.     

Derivatization of small biomolecules for optimized matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a powerful tool for the measurement of low molecular mass compounds of biological interest. The limitations for this method are the volatility of many analytes, possible interference with matrix signals or bad ionization or desorption behavior of the compounds. We investigated the application of well-known and straightforward one-pot derivatization procedures to circumvent these problems. The derivatizations tested allow the measurement and the labeling of alcohols, aldehydes and ketones, carboxylic acids, alpha-ketocarboxylic acids and amines.     

In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide bridged, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF-pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O-glycosylated proteins up to 150 kDa after SDS-PAGE separation.     

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.     

Double cation adduction in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of electron deficient anthraquinone derivatives

Six anthraquinone derivatives were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Clear (pseudo) molecular ions were observed for all the compounds. Interestingly, for some derivatives, strong ions with double cation adduction were also recorded in the positive mode. It is remarkable that all these ions are singly charged. In this work, possible mechanisms for the double cation adduction were investigated and discussed. It appears that the double cation adduction was due to the electron deficient nature of the derivatives, and formed by taking up two singly charged cations and one electron. Substituents on the anthraquinone ring were found to have a significant effect on the double cation adduction. In contrast, no considerable influence of the acidity of MALDI matrix/solution was observed, even on the double proton adduction. Furthermore, it was demonstrated that double cation adduction might occur in the MALDI gas-phase plume. In addition to the anthraquinones, three more electron deficient compounds of different types, i.e. a perylene bisimide derivative (PB), 3,7-decanoylamino-4,8-dihydrobenzo[1,2-b:4,5-b']dithiophene-4,8-dione (TQ) and 6,6-phenyl C61-butyric acid methyl ester (PCBM), were also analyzed with MALDI TOF MS. The results indicate that the 'abnormal' double cation adduction might be a 'normal' phenomenon in the MALDI TOF MS analysis of many electron deficient compounds.     

Radical cation formation in characterization of novel C3-symmetric disks and their precursors by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Four C3-symmetrical tris(dipeptide) disks and their precursors were characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The C3-symmetrical disks were based on a benzene-1,3,5-triscarboxamide core extended by oligopeptides with trialkoxyanilide tails. The results indicate that MALDI TOF MS is a powerful and straightforward analytical technique for characterizing C3-symmetrical disks and their precursors. Clear (pseudo)-molecular ion peaks could readily be identified. It is remarkable that strong radical ion signals were observed for all the compounds, including the anilines that were expected to be protonated prior to laser irradiation using acidic MALDI matrixes. Possible mechanisms for radical ion formation were investigated with the employment of radical scavengers, with various matrixes and with direct laser desorption/ionization (LDI). Most likely the radicals are formed by losing one electron from the aniline nitrogen and stabilized by conjugation through the phenyl ring. It appears that direct photo/thermal ionization of analytes is an important route for the radical ion formation of the compounds with trialkoxy aniline/anilide groups.     

Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibration

Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de-N-glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of +/-0.12 and +/-0.022%, respectively. The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT-III, namely AT-III(alpha) and AT-III(beta), were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135). After exhaustive de-N-glycosylation (by means of PNGase F) of the alpha- and beta-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (-0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N- and O-glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous gamma-carboxyglutamic acids). MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N-glycans.     

Protein secondary structure and stability determined by combining exoproteolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In the post-genomic era, several projects focused on the massive experimental resolution of the three-dimensional structures of all the proteins of different organisms have been initiated. Simultaneously, significant progress has been made in the ab initio prediction of protein three-dimensional structure. One of the keys to the success of such a prediction is the use of local information (i.e. secondary structure). Here we describe a new limited proteolysis methodology, based on the use of unspecific exoproteases coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to map quickly secondary structure elements of a protein from both ends, the N- and C-termini. We show that the proteolytic patterns (mass spectra series) obtained can be interpreted in the light of the conformation and local stability of the analyzed proteins, a direct correlation being observed between the predicted and the experimentally derived protein secondary structure. Further, this methodology can be easily applied to check rapidly the folding state of a protein and characterize mutational effects on protein conformation and stability. Moreover, given global stability information, this methodology allows one to locate the protein regions of increased or decreased conformational stability. All of this can be done with a small fraction of the amount of protein required by most of the other methods for conformational analysis. Thus limited exoproteolysis, together with MALDI-TOF MS, can be a useful tool to achieve quickly the elucidation of protein structure and stability.     

Study of synthetic aliphatic copolyamides by time-of-flight matrix assisted laser desorption/ionization mass spectrometry

Synthetic copolyamides based on aliphatic diamines (1,3-propanediamine and 1,4-butanediamine) and dichlorides of aliphatic carboxylic acids (adipic and sebacic acid dichlorides) were investigated using time-of-flight matrix assisted laser desorption/ionization mass spectrometry. Their mass spectra showed peaks for cationized (Na⁺ and K⁺) and protonated (less intense peaks) oligomers with NH₂-NH₂, NH₂-COOH, or COOH-COOH end groups. No cyclic oligomers were detected in the samples. The compositions of oligomers were determined, and the relative reactivities of homologous comonomers in polycondensation were estimated. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1320–1324, July, 2007.     

Studies on extracting solutions of endohedral rare-earth metallofullerenes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Thirteen extracting solutions of rare-earth metallofullerenes containing La,Ce,Pr,Nd Sm,Eu,Gd,Tb,Dy,Ho,Er,Tm and Yb respectively have been investigated by means of matrix-assisted laser desorpuon/ ionization time-of-flight mass spectrometry.The influences of the positive-ion/negative-ion mode,laser intensity,ma trix and mass discrimination to the analytical results are studied,based on which the optimal analytical conditions have been determined.The results show that the extracting solutions contain large quantities of rare-earth metallofullerenes besides empty fullerenes.On the basis of comparing their relative intensities,the different structure stabilities and solubilities of metallofullerenes with different rare-earth metals encapsulated into the fullerene cages,as well as some possible reasons to those differences,are discussed.     

Fragmentation characteristics of peptide-metal ion adducts under matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometric conditions

Fragmentation reactions of sodium-cationized enkephalin peptides generated by matrix-assisted laser desorption/ionization were studied using post-source decay (PSD) with a reflectron time-of-flight mass spectrometer. Several matrices and analyte-matrix sample preparation methods were evaluated for high-intensity ion currents that could last for the entire PSD analysis. A triple dried-droplet sample preparation procedure with 2,5-dihydroxybenzoic acid as the matrix was found to yield abundant longer-lasting ion signals of the peptide-Na(+) ion adducts. The principal decay product of these adduct ions is the [b(n-1) + Na + OH](+) ion, which provides an unambiguous identification of the C-terminal residue of a peptide. In some peptides, the loss of a second residue from the C-terminus is also observed. No other sequence-specific ions were observed.     

基质辅助激光解吸电离飞行时间质谱研究2: 四硫 富瓦烯化合物

采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),对四硫富瓦烯化合物进行质谱表征。在所用的实验条件下,样品很容易解吸电离生成单电荷分子离子,得到单同位素分辨的质谱图。26种实际样品的质谱分析结果表明;MALDI-TOF-MS可以比其它质谱方法更有效、更方便地用于此类化合物的质谱分析,解决了此类化合物不易进行质谱鉴定的难题。     

A novel sample preparation method of matrix-assisted laser desorption/ionization time of flight mass spectrometry for polystyrene

A novel sample preparation method of matrix-assisted laser desorption/ionization mass spectrometry for polystyrene was reported. Compared to the conventional dried-droplet method, the efficiency of ionization and signal intensity of mass spectra were improved. The mechanism was also analyzed.     

High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 M NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/-0.01-0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0 kDa), 340 ng for glyceraldehyde-3-phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.     

Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ionization and laser desorption/ionization on porous silicon mass spectrometry

Cysteine sulfonic acid-containing peptides, being typical acidic peptides, exhibit low response in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, matrix conditions and the effect of diammonium hydrogencitrate (DAHC) as additive were investigated for ionization of cysteine sulfonic acid-containing peptides in MALDI. A matrix-free ionization method, desorption/ionization on porous silicon (DIOS), was also utilized to evaluate the effect of DAHC. When equimolar three-component mixtures of peptides carrying free cysteine, cysteine sulfonic acid, and carbamidomethyl cysteine were measured by MALDI using a common matrix, alpha-cyano-4-hydroxycinnamic acid (CHCA), no signal corresponding to cysteine sulfonic acid-containing peptide could be observed in the mass spectrum. However, by addition of DAHC to CHCA, the peaks of cysteine sulfonic acid-containing peptides were successfully observed, as well as when using 2,4,6-trihydroxyacetophenone (THAP) and 2,6-dihydroxyacetophenone with DAHC. In the DIOS mass spectra of these analytes, the use of DAHC also enhanced the peak intensity of the cysteine sulfonic acid-containing peptides. On the basis of studies with these model peptides, tryptic digests of oxidized peroxiredoxin 6 were examined as a complex peptide mixture by MALDI and DIOS. In MALDI, the peaks of cysteine sulfonic acid-containing peptides were observed when using THAP/DAHC as the matrix, but this was not so with CHCA. In DIOS, the signal from cysteine sulfonic acid-containing peptides was suppressed; however, the use of DAHC significantly enhanced the signal intensity with an increase in the number of observed peptides and increased signal-to-noise ratio in the DIOS spectra. The results show that DAHC in the matrix or on the DIOS chip decreases discrimination and suppression effects in addition to suppressing alkali-adduct ions, which leads to a beneficial effect on protonation of peptides containing cysteine sulfonic acid.     

Mesoporous tungsten titanate as matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of biomolecules

In this paper, mesoporous tungsten titanate (WTiO) with different nano-pore structures was utilized as matrix for the analysis of short peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Effect of characteristic features of mesoporous matrices on laser desorption/ionization process was investigated. Experiments showed that the ordered two-dimensional and three-dimensional mesoporous matrices were superior in performance to the non-ordered WTiO matrix. The dramatic enhancement of signal sensitivity by the ordered mesoporous matrices can be reasonably attributed to the ordered structure, which facilitated the understanding on structure-function relationship in mesoporous cavity for laser desorption process of adsorbed biomolecules. With the ordered mesoporous matrix, the short peptides are successfully detected. The presence of trace alkali metal salt effectively increased the analyte ion yields and the MALDI-TOFMS using the inorganic mesoporous matrices displayed a high salt tolerance. The developed technique also showed a satisfactory performance in peptide-mapping and amino-acid sequencing analysis.