The disulfide bond pattern of salmon egg lectin 24K from the Chinook salmon Oncorhynchus tshawytscha

The disulfide bonds in the galactose-specific lectin SEL 24K from the egg of the Chinook salmon Oncorhynchus tshawytscha were determined by mass spectrometry. Four predictive in silico tools were used to determine the oxidation state of cysteines in the sequence and possible location of the disulfide bonds. A combination of tryptic digestion, HPLC separation, and chemical modifications were used to establish the location of seven disulfide bonds and one pair of free cysteines. After proteolysis, peptides containing one or two disulfide bonds were identified by reduction and mass spectral comparison. MALDI mass spectrometry was supported by chemical modification (iodoacetamide) and in silico digestion. The assignments of disulfide bonds were further confirmed by mass spectral fragmentation studies including in-source dissociation (ISD) and collision-induced dissociation (CID). The experimentally determined disulfide bonds and free Cys residues were only partially consistent with those generated by several automated public-domain algorithms.     

The essence of DNA sample preparation for MALDI mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important analytical technique in nucleic acid research. MALDI is used for quality control of oligonucleotides as well as for analyzing DNA markers. Sample preparation of nucleic acids is crucial for obtaining high-quality mass spectra. Sample purity, solvent content, suitable matrices, and substrate surfaces, as well as laboratory conditions affect spectra quality. This review presents essential information with regard to sample preparation, DNA modification chemistry, and DNA purification, along with a discussion of instrumental advances, which facilitate and extend the applicability of MALDI in genomics.     

The essence of DNA sample preparation for MALDI mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important analytical technique in nucleic acid research. MALDI is used for quality control of oligonucleotides as well as for analyzing DNA markers. Sample preparation of nucleic acids is crucial for obtaining high-quality mass spectra. Sample purity, solvent content, suitable matrices, and substrate surfaces, as well as laboratory conditions affect spectra quality. This review presents essential information with regard to sample preparation, DNA modification chemistry, and DNA purification, along with a discussion of instrumental advances, which facilitate and extend the applicability of MALDI in genomics.     

Mass spectrometric strategy for primary structure determination of N-terminally blocked peptides

The mass spectrometric strategy including three steps is presented for primary structure determination of the N-terminally blocked peptides. First, the C-terminal sequencing is performed by using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry coupled with carboxypeptidase Y digestion. Then, the peptide is cleaved according to the obtained C-terminal sequence information and the resulting peptides are identified by mass spectrometry and Edman degradation after fractionation by reverse-phase chromatography. Finally, the N-terminal fragment is sequenced by tandem mass spectrometry. The strategy was successfully applied to the sequence determination of two novel N-terminally blocked peptides named EAFP1 and EAFP2.     

Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry

The cloning, sequencing and high-level expression of the gene encoding extracellular lipase from Streptomyces rimosus R6-554W have been recently described, and the primary structure of this gene product was deduced using a bioinformatic approach. In this study, capillary electrophoresis-on-the-chip and mass spectrometry were used to characterize native and overexpressed extracellular lipase protein from S. rimosus . The exact molecular mass of the wild-type and the overexpressed lipase, determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, were in excellent agreement (Deltam=0.11 Da and Deltam=0.26 Da, respectively) with a value of 24165.76 Da calculated from the structure deduced from the nucleotide sequence, considering the mature enzyme with all six cysteines forming disulfide bridges. The primary structure derived from the nucleotide sequence was completely verified using a combination of tryptic digestion and formic acid cleavage of the protein, followed by peptide mass fingerprinting. Selected peptides were further investigated by MALDI low-energy collision-induced dissociation hybrid tandem mass spectrometry, allowing the unambiguous determination of their predicted amino acid sequence. No post-translational modifications of mature S. rimosus lipase were detected. Comparison of the peptide mass fingerprints from the reduced and non-reduced overexpressed enzyme unequivocally revealed three intramolecular disulfide bonds with the following linkages: C27-C52, C93-C101 and C151-C198.     

Highly informative proteome analysis by combining improved N-terminal sulfonation for de novo peptide sequencing and online capillary reverse-phase liquid chromatography/tandem mass spectrometry

Recently, various chemical modifications of peptides have been incorporated into mass spectrometric analyses of proteome samples, predominantly in conjunction with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), to facilitate de novo sequencing of peptides. In this work, we investigate systematically the utility of N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate (SPITC) for proteome analysis by capillary reverse-phase liquid chromatography/tandem mass spectrometry (cRPLC/MS/MS). The experimental conditions for the sulfonation were carefully adjusted so that SPITC reacts selectively with the N-terminal amino groups, even in the presence of the epsilon-amino groups of lysine residues. Mass spectrometric analyses of the modified peptides by cRPLC/MS/MS indicated that SPITC derivatization proceeded toward near completion under the experimental conditions employed here. The SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra. Combining SPITC derivatization and cRPLC/MS/MS analyses facilitated the acquisition of sequence information for lysine-terminated tryptic peptides as well as arginine-terminated peptides without the need for additional peptide pretreatment, such as guanidination of lysine amino group. This process alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI MS experiments. We will discuss the utility of the technique as a viable method for proteome analyses and present examples of its application in analyzing samples having different levels of complexity.     

Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 from Momordica charantia

A codon-optimized recombinant ribonuclease, MC1 is characterized for its uridine-specific cleavage ability to map nucleoside modifications in RNA. The published MC1 amino acid sequence, as noted in a previous study, was used as a template to construct a synthetic gene with a natural codon bias favoring expression in Escherichia coli. Following optimization of various expression conditions, the active recombinant ribonuclease was successfully purified as a C-terminal His-tag fusion protein from E. coli [Rosetta 2(DE3)] cells. The isolated protein was tested for its ribonuclease activity against oligoribonucleotides and commercially available E. coli tRNA^{Tyr I}. Analysis of MC1 digestion products by ion-pairing reverse phase liquid-chromatography coupled with mass spectrometry (IP-RP-LC-MS) revealed enzymatic cleavage of RNA at the 5′-termini of uridine and pseudouridine, but cleavage was absent if the uridine was chemically modified or preceded by a nucleoside with a bulky modification. Furthermore, the utility of this enzyme to generate complementary digestion products to other common endonucleases, such as RNase T1, which enables the unambiguous mapping of modified residues in RNA is demonstrated.     

Bacterial genotyping by 16S rRNA mass cataloging

Background  

It has recently been demonstrated that organism identifications can be recovered from mass spectra using various methods including base-specific fragmentation of nucleic acids. Because mass spectrometry is extremely rapid and widely available such techniques offer significant advantages in some applications. A key element in favor of mass spectrometric analysis of RNA fragmentation patterns is that a reference database for analysis of the results can be generated from sequence information. In contrast to hybridization approaches, the genetic affinity of any unknown isolate can in principle be determined within the context of all previously sequenced 16S rRNAs without prior knowledge of what the organism is. In contrast to the original RNase T₁ cataloging method, when digestion products are analyzed by mass spectrometry, products with the same base composition cannot be distinguished. Hence, it is possible that organisms that are not closely related (having different underlying sequences) might be falsely identified by mass spectral coincidence. We present a convenient spectral coincidence function for expressing the degree of similarity (or distance) between any two mass-spectra. Trees constructed using this function are consistent with those produced by direct comparison of primary sequences, demonstrating that the inherent degeneracy in mass spectrometric analysis of RNA fragments does not preclude correct organism identification.     

N-terminus determination: FAD and NADP binding domain mapping of hog liver flavin-containing monooxygenase by tandem mass spectrometry

Highly purified hog liver flavin-containing monooxygenase was sequentially denatured, reduced, carboxymethylated, and digested with endoproteinase Glu-C. The purified peptides were subjected to mass spectrometric analysis and the amino acid sequence of selected fragments was determined by tandem mass spectrometry. The amino acid sequence of the first 12 residues of the N-terminus was: Ac-Ala-Lys-Arg-Val-Ala-Ile-Val-Gly-Ala-Gly-Val-Ser-Gly. The amino acid sequence determined for another peptide was: Lys-Ser-Val-Leu-Val-Val-Gly-Met-Gly-Asn-Ser-Gly-Thr-Asp-Ile-Ala-Val-Glu. The results provide direct evidence for the structure of the N-terminal modification of the protein and for the existence of the FAD and NADP binding domains of Gly-X-Gly-X-X-Gly.     

Mass spectrometry-based sequencing of protein C-terminal peptide using α-carboxyl group-specific derivatization and COOH capturing

An approach to mass spectrometry (MS)-based sequence analysis of selectively enriched C-terminal peptide from protein is described. This approach employs a combination of the specific derivatization of α-carboxyl group (α-COOH), enzymatic proteolysis using endoproteinase GluC, and enrichment of C-terminal peptide through the use of COOH-capturing material. Highly selective derivatization of α-COOH was achieved by a combination of specific activation of α-COOH through oxazolone chemistry and amidation using 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine). This amine component was used to simplify fragmentation in tandem mass spectrometry (MS/MS) measurement, which facilitated manual sequence interpretation. The peptides produced after GluC digestion were then treated with a COOH scavenger to enrich the C-terminal peptide that is only devoid of COOH groups, and the obtained C-terminal peptide was readily sequenced by matrix-assisted laser desorption/ionization (MALDI)-MS/MS due to the TMPP mass tag.     

A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program

Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described.     

Identification of tyrosine 204 as the photo-cross-linking site in the DNA-EcoRI DNA methyltransferase complex by electrospray ionization mass spectrometry

We describe a highly sensitive strategy combining laser-induced photo-cross-linking and HPLC-based electrospray ionization mass spectrometry to identify amino acid residues involved in protein-DNA recognition. The photoactivatible cross-linking thymine isostere, 5-iodoracil, was incorporated at a single site within the sequence recognized by EcoRI DNA methyltransferase (GAATTC). UV irradiation of the DNA-protein complex at 313 nm results in a >60% cross-linking yield. SDS-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the covalent cross-linked complex. The total mass is consistent with covalent bond formation between one strand of DNA and the protein with 1:1 stoichiometry. Protease digestion of the cross-linked complex yields several peptide-DNA adducts that were purified by anion-exchange column chromatography. A combination of mass spectrometric analysis and amino acid sequencing revealed that tyrosine 204 was cross-linked to the DNA. Electrospray mass spectrometric analysis of the peptide-nucleoside adduct confirmed this assignment. Tyrosine 204 resides in a peptide motif previously thought to be involved in AdoMet binding and methyl transfer. Thus, amino acids within loop segments but outside of "DNA binding" motifs can be critical to DNA recognition. Our method provides an accurate characterization of picomole quantities of DNA-protein complexes.     

Identification of three novel peptides isolated from the venom of the neotropical social wasp Polistes major major.

Three novel peptides designated as PMM1, PMM2, and PMM3 were isolated and characterized from the venom of the social wasp Polistes major major, one of the most common wasps in the Dominican Republic. By Edman degradation, and MALDI-TOF and ESI-QTOF mass spectrometry, the primary sequences of these peptides were established as follows: PMM1, H-Lys-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH (1357.77 Da); PMM2, H-Ile-Asn-Trp-Lys-Lys-Ile-Ala-Ser-Ile-Gly-Lys-Glu-Val-Leu-Lys-Ala-Leu-NH2 (1909.19 Da); and PMM3, H-Phe-Leu-Ser-Ala-Leu-Leu-Gly-Met-Leu-Lys-Asn-Leu-NH2 (1317.78 Da). The suggested sequences were confirmed by MS analysis of peptide fragments obtained by enzymatic digestion. The peptide PMM1 is a lysyl-arginyl-Thr(6)-bradykinine that belongs to the wasp kinins group. The sequence of the PMM2 peptide is unique; it resembles somewhat the tetradecapeptide amides of the mastoparan group; however, the chain is extended by three additional amino acid residues. The sequence of PMM3 dodecapeptide is homologous to the peptides of the wasp chemotactic group.     

C-terminal sequence determination of peptides degraded with carboxypeptidases of different specificities and analyzed by 252-Cf plasma desorption mass spectrometry

Identification of the truncated peptides by plasma desorption mass spectrometry in C-terminal sequence determination with carboxypeptidases offers several advantages over analysis of the liberated amino acids. It is possible to perform in situ digestion of a nitrocellulose-bound sample already used for molecular weight determination and thus obtain sequence information without further sample consumption. In time-course analysis the analytical information, although not obtained in real time, is sufficiently rapid to adjust the digestion conditions. There is no need for quantitation because the identification is based on molecular weight differences. Sensitivity in the low picomole range is obtainable. The digestion of a number of peptides (900-3500 Da) with carboxypeptidase Y and MII has been monitored. It was found that successive use of the enzymes or use of a mixture of the enzymes was often advantageous. The sequence of up to 10 residues from the C-terminus has been determined for the peptides studied.     

Identities and phylogenetic comparisons of posttranscriptional modifications in 16 S ribosomal RNA from Haloferax volcanii

Small subunit (16 S) rRNA from the archaeon Haloferax volcanii, for which sites of modification were previously reported, was examined using mass spectrometry. A census of all modified residues was taken by liquid chromatography/electrospray ionization-mass spectrometry analysis of a total nucleoside digest of the rRNA. Following rRNA hydrolysis by RNase T(1), accurate molecular mass values of oligonucleotide products were measured using liquid chromatography/electrospray ionization-mass spectrometry and compared with values predicted from the corresponding gene sequence. Three modified nucleosides, distributed over four conserved sites in the decoding region of the molecule, were characterized: 3-(3-amino-3-carboxypropyl)uridine-966, N(6)-methyladenosine-1501, and N(6),N(6)-dimethyladenosine-1518 and -1519 (all Escherichia coli numbering). Nucleoside 3-(3-amino-3-carboxypropyl)uridine, previously unknown in rRNA, occurs at a highly conserved site of modification in all three evolutionary domains but for which no structural assignment in archaea has been previously reported. Nucleoside N(6)-methyladenosine, not previously placed in archaeal rRNAs, frequently occurs at the analogous location in eukaryotic small subunit rRNA but not in bacteria. H. volcanii small subunit rRNA appears to reflect the phenotypically low modification level in the Crenarchaeota kingdom and is the only cytoplasmic small subunit rRNA shown to lack pseudouridine.     

Isoforms of a cuticular protein from larvae of the meal beetle, Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis.

Simultaneous sequencing, using a combination of mass spectrometry and Edman degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants.