Tryptic Digest Fragments Research Articles

Investigating the suitability of high-resolution mass spectrometry for newborn screening: identification of hemoglobinopathies and β-thalassemias in dried blood spots.

A fast and reliable method for the determination of hemoglobinopathies and thalassemias by high-resolution accurate mass spectrometry (HRAM/MS) is presented. The established method was verified in a prospective clinical study (HRAM/MS vs. high-pressure liquid chromatography [HPLC]) of 5335 de-identified newborn samples from the Hamburg area. The analytical method is based on a dual strategy using intact protein ratios for thalassemias and tryptic digest fragments for the diagnosis of hemoglobinopathies. Due to the minimal sample preparation and the use of flow injection, the assay can be considered as a high-throughput screening approach for newborn screening programs (2 min/sample). Using a simple dried blood spot (DBS) extraction (tryptic digest buffer), the following results were obtained: (1) a carrier incidence of 1:100 newborns (35 FAS, nine FAC, eight FAD and two FAE), and (2) no homozygous affected patient was detected. Using the HRAM/MS protocol, an unknown Hb mutation was identified and confirmed by genetic testing. In addition to greater specificity toward rare mutations and β-thalassemia, the low price/sample (1-2€) as well as an automated data processing represent the major benefits of the described HRAM/MS method.

Conlinin in flaxseed (Linum usitatissimum L.) gum and its contribution to emulsification properties

Flaxseed gum (FG) is a mixture of natural polysaccharide and protein derived from flaxseed (Linum usitatissimum L.) that has potential for thickening foods, stabilizing emulsions, and gelling solutions. The composition and identities of proteins in FG have never been reported. In this study, gum prepared from whole flaxseed was deglycosylated by treating it with trifluoromethanesulfonic acid (TFMS). The resultant proteins were separated by 2D-gel electrophoresis. The major protein spots with estimated molecular weights (MW) of 10–11 kDa and 11–12 kDa were excised, digested with trypsin, and analyzed using matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Peptide MS of tryptic digestion fragments was compared to MS of peptides from gene models available through National Center for Biotechnology Information (NCBI) database. Fragments consistent with the seed storage protein conlinin, the low-molecular-mass 2S storage flaxseed protein, were identified as the major spot constituents. Emulsification properties of FG were determined before and after protease hydrolysis with emulsion activity index (EAI) and emulsion stability (ES) as indicators. Both EAI and ES decreased from 98.7 ± 5.4 to 59.9 ± 3.2 m2/g and from 66.4 ± 1.1 to 42.1 ± 2.0%, respectively, after protease treatment. Conlinin is the major protein associated with FG and it plays a fundamental role in determining FG emulsification properties.

Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase fromPseudomonassp. strain ADP

The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Å resolution and adopted space group P21, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, β=106.6°.

Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with (13)C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia.

Calmodulin Wraps around Its Binding Domain in the Plasma Membrane Ca2+ Pump Anchored by a Novel 18-1 Motif

Using solution NMR spectroscopy, we obtained the structure of Ca(2+)-calmodulin (holoCaM) in complex with peptide C28 from the binding domain of the plasma membrane Ca(2+)-ATPase (PMCA) pump isoform 4b. This provides the first atomic resolution insight into the binding mode of holoCaM to the full-length binding domain of PMCA. Structural comparison of the previously determined holoCaM.C20 complex with this holoCaM.C28 complex supports the idea that the initial binding step is represented by (holoCaM.C20) and the final bound complex by (holoCaM.C28). This affirms the existing multi-step kinetic model of PMCA4b activation by CaM. The complex exhibits a new binding motif in which holoCaM is wrapped around helical C28 peptide using two anchoring residues from the peptide at relative positions 18 and 1. The anchors correspond to Phe-1110 and Trp-1093, respectively, in full-length PMCA4b, and the peptide and CaM are oriented in an anti-parallel manner. This is a greater sequence distance between anchors than in any of the known holoCaM complexes with a helical peptide. Analysis of the geometry of holoCaM-peptide binding for the cases where the target peptide adopts an alpha(D)-helix with its anchors buried in the main hydrophobic pockets of the two CaM lobes establishes that only relative sequential positions of 10, 14, 17, and 18 are allowed for the second anchor.

In Vivo Residue-specific Histone Methylation Dynamics

Methylation of specific histone residues is capable of both gene activation and silencing. Despite vast work on the function of methylation, most studies either present a static snapshot of methylation or fail to assign kinetic information to specific residues. Using liquid chromatography-tandem mass spectrometry on a high-resolution mass spectrometer and heavy methyl-SILAC labeling, we studied site-specific histone lysine and arginine methylation dynamics. The detection of labeled intermediates within a methylation state revealed that mono-, di-, and trimethylated residues generally have progressively slower rates of formation. Furthermore, methylations associated with active genes have faster rates than methylations associated with silent genes. Finally, the presence of both an active and silencing mark on the same peptide results in a slower rate of methylation than the presence of either mark alone. Here we show that quantitative proteomic approaches such as this can determine the dynamics of multiple methylated residues, an understudied portion of histone biology.

Photoaffinity labeling and mutational analysis of 24-C-sterol methyltransferase defines the AdoMet binding site.

Photolabeling and site-directed mutagenesis were performed on recombinant Saccharomyces cerevisiae 24-C-sterol methyltransferase (SMT) to elucidate the location and role of active site residues involved in AdoMet binding and catalysis. Bioinformatic analysis of the SMT revealed a ten amino acid segment, conserved between L124 and P133, associated with the Rossmann-like fold belonging to AdoMet-dependent methyltransferases. Irradiation of the SMT in the presence of [methyl-3H3]AdoMet directly photolabeled the protein. The specificity of photolabeling was demonstrated by inactivation experiments with structural analogs of AdoMet, including sinefungin. Trypsin digestion of the [methyl-3H3]AdoMet photolabeled Erg6p afforded a single radioactive band in SDS-PAGE gel of 4 kDa. HPLC purification of this material generated a single radioactive fraction. The corresponding 3H-AdoMet-peptide adduct was subjected to Edman sequencing and the first fifteen residues gave a sequence Gly120-Asp-Leu-Val-Leu-Asp-Val-Gly-Cys-Gly-Val-Gly-Gly-Pro-Ala134 that contained the predicted AdoMet binding site. Amino acid residues in the tryptic digest fragment considered to bind covalently with the AdoMet at Asp125, Cys128, Pro133 and Tyr153 were replaced with leucine and analyzed kinetically and by photolabeling inactivation experiments. The results indicate that one or both of Cys128 and Pro133 are covalently bound to AdoMet.

A Novel Site-Directed Affinity Reagent for Cross-Linking Human Hemoglobin: Bis[2-(4-phosphonooxyphenoxy)carbonylethyl]phosphinic Acid

Bis[2-(4-phosphonooxyphenoxy)carbonylethyl]phosphinc acid (BPPCEP) was prepared and evaluated as a site-directed affinity reagent for cross-linking human hemoglobin. It was synthesized in four steps starting from 4-benzyloxyphenol and was converted to its pentasodium salt so as to afford efficient cross-linking in an aqueous medium. The reagent was found to specifically cross-link human hemoglobin A(0) in the beta-cleft chains under oxygenated reaction conditions at neutral pH. The amino acid residues involved in the cross-linking were determined by mass spectral analyses of tryptic digest fragments of cross-linked hemoglobin, employing a MALDI-TOF mass spectrometer. The MS analyses suggested that the most likely amino acids involved in the cross-links are Val-1 or Lys-82 present on one of the beta subunits and Lys-82 or Lys-144 on the other. Molecular modeling studies performed on the reagent-HbA(0) complex corroborated the conclusions reached by MALDI-MS analyses. The oxygen equilibrium measurements of the three major BPPCEP-cross-linked Hb products, isolated and purified by preparative cation exchange chromatography, exhibited oxygen affinity (P(50)) values of 14.5, 12.1, and 15.5 Torr as compared with the P(50) of 13.1 Torr for cell-free hemoglobin. The oxygen-binding cooperativity of the modified products, as determined by the Hill coefficient generated from the Hill plots of the respective P(50) values, coupled with the absence of sigmoidal shape of the O(2) equilibrium curves, was considerably lower than that of the native hemoglobin.

Vibrational spectroscopic studies of solid recombinant bovine growth hormone and related growth hormone analogs

Infrared and Raman spectra have been obtained for lyophilized recombinant bovine growth hormone (r-bGH), partially reduced, and completely reduced r-bGH, plus a tryptic digest fragment of r-bGH. Amide I and II data indicate r-bGH to have substantial helical character. Partially reduced r-bGH, in which the carboxyl terminal disulfide bridge (residues 181, 189) has been cleaved, has slightly less helical content than r-bGH. The spectral data indicate that breaking the carboxyl terminal cystine link produces only localized structural alterations. The additional cleavage of the second disulfide bridge (residues 53 164) leads to a further decrease in helix content, accompanied by increases in β-sheet and disordered structures. A tryptic digest r-bGH fragment (residues 96–133), which contains a small amount of biological activity (≈10%), has predominantly helical structure.

Molecular Cloning and Functional Expression of Mannitol-1-phosphatase from the Apicomplexan Parasite Eimeria tenella

A metabolic pathway responsible for the biosynthesis and utilization of mannitol is present in the seven species of Eimeria that infect chickens, but is not in the avian host. Mannitol-1-phosphatase (M1Pase), a key enzyme for mannitol biosynthesis, is a highly substrate-specific phosphatase and, accordingly, represents an attractive chemotherapeutic target. Amino acid sequence of tryptic peptides obtained from biochemically purified Eimeria tenella M1Pase was used to synthesize degenerate oligonucleotide hybridization probes. Using these reagents, a partial genomic clone and full-length cDNA clones have been isolated and characterized. The deduced amino acid sequence of E. tenella M1Pase shows limited overall homology to members of the phosphohistidine family of phosphatases. This limited homology to other histidine phosphatases does, however, include several conserved residues that have been shown to be essential for their catalytic activity. Kinetic parameters of recombinant M1Pase expressed in bacteria are essentially identical to those of the biochemically purified preparation from E. tenella. Moreover, recombinant M1Pase is subject to active site-directed, hydroxylamine-reversible inhibition by the histidine-selective acylating reagent diethyl pyrocarbonate. These results indicate the presence of an essential histidine residue(s) at the M1Pase active site, as predicted for a histidine phosphatase.

Probing the conformation of NhaA, a Na+/H+ antiporter from Escherichia coli, with trypsin.

One of the most striking features of NhaA, an Escherichia coli Na+/H+ antiporter, is its extreme sensitivity to pH. The activity of NhaA increases 2000-fold between pH 6.5 and 8.5. In this work, we investigated whether the activation of NhaA by pH is accompanied by conformational changes which can be detected using trypsin as a probe. We have found that NhaA is susceptible to proteolytic digestion at the pH range where it is activated, suggesting that these two events may be related; at alkaline pH, the protein becomes active and adopts an "open" conformational state in which more domains are exposed to the enzyme. This idea was further supported by results from two mutants of NhaA in which His-225, a residue involved in pH sensing, has been replaced by either Arg or Asp. The mutant H225R is activated at more acidic pH values, while H225D at more alkaline pH. In accordance with the results described for the wild-type protein, H225R was susceptible to digestion by trypsin at the pH at which it undergoes main activation. NhaA has many potential tryptic cleavage sites. However, analysis of the tryptic digestion fragments suggests that at alkaline pH, the protein is exposed to cleavage mainly at hydrophilic loops 6, 7, and 8. Thus, upon activation, NhaA appears to undergo a change in conformation that is reflected in specific regions of the protein.

Ionic Reactivity of Mastoparan and its Tryptic Digest Fragments Under Fast‐atom Bombardment

Gas-phase reactivity of the native mastoparan from Vespula lewissi and that of its tryptic digest fragments are studied giving directly sequence information and discrimination between isobaric residues. © 1997 John Wiley & Sons, Ltd.

Ionic Reactivity of Mastoparan and its Tryptic Digest Fragments Under Fast-atom Bombardment

Gas-phase reactivity of the native mastoparan from Vespula lewissi and that of its tryptic digest fragments are studied giving directly sequence information and discrimination between isobaric residues. © 1997 John Wiley & Sons, Ltd.

Red algal LHC I genes have similarities with both Chl a/b- and a/c-binding proteins: A 21 kDa polypeptide encoded by LhcaR2 is one of the six LHC I polypeptides

Polypeptides from the PS I holocomplex of the red alga P. cruentum, purified for microsequencing, confirmed that six LHC I polypeptides from SDS-PAGE are distinct apoproteins. Analysis of a cDNA clone, designated as LhcaR2, from a cDNA library, indicates that it shares major structural features with the recently cloned first red algal gene LhcaR1. The LhcaR2 is believed to encode the 21.0 kDa polypeptide of the LHC I complex from comparison of the deduced amino acid sequence and the microsequences of several tryptic digest fragments from the isolated polypeptide. As in chlorophytic and chromophytic LHCs, the essential residues for Chl-binding and helix stabilization in helices 1 and 3 are highly conserved. Relatedness between rhodophytes and the chlorophytes is also inferred from sequence conservation in the N-flanking regions of helices 1 and 3. Conversely, helix 2 exhibited the highest similarity between LHC sequences of Chl a/c-binding chromophytes and the Chl a-binding rhodophytes, with 11 of 22 residues identical or conservatively substituted. Moreover, whereas in chlorophytes, the Q and E Chl-binding residues are separated by seven amino acid residues, they are always separated by 8 residues in rhodophytes and chromophytes. Superimposition of the predicted LhcaR2 sequence with the LHC II model [Kuhlbrandt et al. (1994) Nature 367: 614–621] shows the same structural features except shortened connecting sequence between helices 1 and 2 on the lumenal side. The chimeric nature of rhodophyte genes, with both chromophytic and chlorophytic features, leads to the suggestion that they reflect attributes of an intermediate stage in LHC apoprotein evolution.

Expression of Monovalent Fragments Derived from a Human IgM Autoantibody in E. Coli. The Input of the Somatically Mutated CDR1/CDR2 and of the CDR3 into Antigen Binding Specificity

A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and V lambdaIII genes. To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment obtained by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFcl.l. fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody. The expression vectors contained at the 3' end either a (His) 6 motif allowing purification on Ni 2+-agarose or a e-m yc tag for specifically detecting the expression products by a murine monoclonal antibody. Western blotting and ELISA analyses of the expression

Cross-species identification of proteins separated by two-dimensional gel electrophoresis using matrix-assisted laser desorption ionisation/time-of-flight mass spectrometry and amino acid composition.

Amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry were used to identify nine of twelve proteins originally separated by two-dimensional electrophoresis and derived from an organism poorly defined at the molecular level (Spiroplasma melliferum). Two of three unidentified proteins appeared to be novel. The percentage amino acid composition and the molecular mass of peptide fragments generated by tryptic digestion were used to search the PIR/SWISS-PROT and MOWSE databases respectively. Lists of candidate proteins were independently generated and ranked from data obtained by both methods. A putative identification was allocated when a single candidate protein appeared in both lists of computer-generated rankings. Results were verified using N-terminal protein microsequencing. The combined use of amino acid composition and MALDI-TOF mass spectrometry allowed a high degree of confidence to be placed in such identifications because they were based upon homologous data sets of at least 20 parameters (16 amino acids and 4-10 tryptic digest fragments). A further two parameters, estimated M(r) and, to a lesser extent, pI, were also used to reinforce this measure of confidence. Ranking of candidate proteins by one method alone could lead to false identification. Both techniques can process large numbers of samples rapidly. In light of the increasing number of entries in both gene and protein databases, this approach is likely to become an essential first step for the characterisation of proteins, particularly across species boundaries.

Improved resolution of tryptic digest fragments from haemoglobin variants using phytic acid in free zone capillary electrophoresis.

Capillary electrophoresis can be applied to the rapid characterization of tryptic digests of proteins. The addition of phytic acid to the separation buffer was found to improve resolution considerably when the technique was applied to differentiate between tryptic digests derived from variant haemoglobins. Moreover, analysis time was of the order of 15 min, which is considerably shorter than that obtained using gradient reversed-phase high-performance liquid chromatography or two-dimensional paper chromatography-electrophoresis.

Determination of the amino acid sequence of apamin by tandem mass spectrometry

A new strategy has been developed for the determination of the amino acid sequence of apamin, a two disulfide-bond neurotoxic oligopeptide. Relative molecular mass up to about 2000 was determined using electrospray mass spectrometry. This technique shows that three basic amino acid residues are present in apamin. A hydrophobic tryptic digest fragment was sequenced using fast-atom bombardment combined with tandem mass spectrometry. But the full sequence has been directly observed using a tandem mass spectrometer: the daughter-ion mass spectrum of protonated molecules of dithiothreitol-reduced apamin gives the sequence.

Free-Solution Capillary Electrophoresis of Tryptic Digest Fragments of a Recombinant Porcine Pro-Growth Hormone Releasing Hormone (2–76)Oh

Abstract The application of free-solution capillary electrophoresis (FSCE) to the separation of peptide fragments produced by tryptic digestion of a new recombinant porcine pro-growth hormone releasing hormone (2–76)OH (rpGHRH) composed of 75 amino acid residues is presented. It was found that 11 digest peptide fragments of rpGHRH could be separated by FSCE using an uncoated fused silica capillary with 0.1 M phosphate buffer, pH 3.3, used as the separating electrolyte. Individual peptide fragments prepared by solid phase synthesis method were spiked into the tryptic digest mixture to verify structure and fragment assignments of the polypeptide in electrophoresis. Optimal separation conditions were obtained by studying the effect of pH in the separating electrolyte and applied voltage. An excellent correlation was found for the electrophoretic migration time, tm, of the digest peptides versus q/MW2/3, where q is the calculated charge, and MW is molecular weight of peptides.

Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase.

An exopolyphosphatase [exopoly(P)ase; EC 3.6.1.11] activity has recently been purified to homogeneity from a mutant strain of Escherichia coli which lacks the principal exopoly(P)ase. The second exopoly(P)ase has now been identified as guanosine pentaphosphate phosphohydrolase (GPP; EC 3.6.1.40) by three lines of evidence: (i) the sequences of five tryptic digestion fragments of the purified protein are found in the translated gppA gene, (ii) the size of the protein (100 kDa) agrees with published values for GPP, and (iii) the ratio of exopoly(P)ase activity to GPP activity remains constant throughout a 300-fold purification in the last steps of the procedure. The enzyme liberates orthophosphate by processive hydrolysis of the phosphoanyhydride bonds of polyphosphate [poly(P)] chains (1000 residues) or by hydrolysis of the 5'-gamma-phosphate of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) to guanosine 5'-diphosphate 3'-diphosphate (ppGpp or "magic spot"). The Km for long-chain poly(P) as a substrate (approximately 0.5 nM) is far lower than that for pppGpp (0.13 mM); the kcat for the poly(P)ase activity is 1.1 s-1 and that for pppGpp hydrolase is 0.023 s-1. These and other findings direct attention to possible functions of poly(P) in the response of E. coli to stresses and deprivations.