Analyses of In Vitro Nonenzymatic Glycation of Normal and Variant Hemoglobins by MALDI-TOF Mass Spectrometry

MALDI-TOF mass spectrometry is used here to differentiate different glycoisoforms of normal and variant hemoglobins (Hbs) in nonenzymatic in vitro glycation. Single, double, and/or multiple glycation of the α-globin, β-globin, and/or γ-globin is observed. Different glycation rates are observed for various Hbs, and the normal Hb A has the slowest rate. Although the Hb A is relatively stable upon condensation with glucose at 37°C, the variants Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego are less stable. In addition, data reveal that the number of glucose attached/Hb molecule (state of glycation) increases with longer incubation time, higher glucose concentration, and higher temperature. The pH dependence of the state of glycation is more complex and varies for different Hbs. Although pH has little effect on the state of glycation for Hb C, Hb E, and Hb Leiden, it increases for Hb A and Hb F upon changing the pH of the solution from phosphate buffer saline (pH 7.4) to carbonate buffer (pH 10). Results obtained in this study could lead to the inference that the linkage of Hbs with glucose occurs in diabetic conditions in vivo (37°C, ∼neutral pH, ∼0.007 M glucose), and the state of glycation is more severe in the individuals who carry abnormal Hbs.     

Mass spectrometry analyses of rat 2b myosin heavy chain isoform

We have separated 2b myosin heavy chain (MyHC) isoform from the rat extensor digitorum longus muscle by SDS-PAGE and analyzed it by two subsequent mass spectrometry techniques. After tryptic digestion, the obtained peptides were identified by Matrix-Assisted Laser Desorption/Ionisation reflectron Time of Flight mass spectrometry (MALDI-TOF MS) and sequenced by liquid chromatography tandem mass spectrometry (ESI LC/MS/MS). The analyzed peptides proportionally covered 30 % of the 2b MyHC isoform sequence. The results suggest that the primary structure is identical with the highest probability to a NCBI database record ref|NP_062198.1|, representing the last updated record of rat 2b isoform. Nonetheless, four peptides carrying amino acid substitution(s) in comparison with the NCBI database record were identified.     

Dual-source mass spectrometer with MALDI-LIT-ESI configuration

A novel linear ion trap (LIT) mass spectrometer with dual matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) ionization sources has been built in the MALDI-LIT-ESI configuration. The design features two independent ion source/ion optical channels connected to opposite ends of a single mass analyzer. The instrument consists of a commercial MALDI-LIT instrument modified by the addition of a home-built vacuum manifold, ion optical system, control electronics, and programming necessary to couple an atmospheric pressure interface to the commercial instrument. In addition to the added ESI functionality, the capabilities of the system also include simultaneous dual-channel ion introduction and analysis and high-duty cycle electronic switching (<1 s) between ion channels. Analytical and ion chemical applications of the dual-source system are explored. One analytical application is the enhanced protein sequence coverage achieved when using both ESI and MALDI to examine a tryptic digest of a six-protein mixture. The differences in the efficiency with which peptides in a mixture are ionized by the two methods give improved sequence coverage when both are applied. Other analytical applications include the use of the ions from one source as intensity or mass standards for the analyte ions from the other. An ion chemistry application involves the use of energy-resolved tandem mass spectrometry (MS/MS) to seek evidence for the generation of isomeric ions from a particular compound using the two ionization methods. A high level of agreement was achieved between the MS/MS spectra recorded under a variety of conditions after ESI and MALDI ionization; this provides evidence of the reproducibility and internal consistency of data from the dual source instrument. However, each of the peptides examined generated identical populations of structures in the two ionization methods under our conditions which are interpreted as involving slow cooling into the most stable minimum on the potential energy surface.     

Study on the synthesis and characterization of peptides containing phosphorylated tyrosine.

Phosphorylation and dephosphorylation are key events in receptor-mediated and post-receptor-mediated signal transduction. Synthetic phosphopeptides have been shown to have dramatic agonist or antagonist effects in several of these signaling pathways. For its 1997 study, the Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group assessed the ability of member laboratories to synthesize phosphotyrosine peptides. Participating laboratories were requested to synthesize and submit the following crude peptide, H-Glu-Asp-Tyr-Glu-Tyr(PO3H2)-Thr-Ala-Arg-Phe-NH2, for evaluation by amino acid analysis, sequence analysis, RP-HPLC, MALDI-TOF and ESI mass spectrometry. Prior to analysis of submitted peptides from ABRF members, the Peptide Synthesis Research Group synthesized and characterized the nonphosphorylated form of the peptide, the doubly phosphorylated form and the peptides singly phosphorylated on either the first or the second tyrosine. These peptide standards were separated easily by HPLC and capillary electrophoresis and the phosphotyrosine was detected readily by Edman degradation sequence analysis. No differences were seen by amino acid analysis and the expected masses were observed by mass spectrometry. The two singly phosphorylated peptides were easily distinguished by MALDI-PSD. Analysis of the peptides submitted from member facilities revealed that all but four of the 33 samples contained the correct product as determined by HPLC and mass spectrometry. HPLC analysis indicated that 20 of the 33 submitted samples contained greater than 75% correct product, five contained less than 50% correct product and four did not contain any correct product. By ESI/MS, an additional singly charged ion at m/z 535.5 was detected in five of the 33 submitted samples; this ion was subsequently shown to represent Ac-TARF-NH2. No correlation was found to exist between coupling time and percentage correct product; however, a correlation may exist between a greater percentage of correct product and the use of non-protected phosphotyrosine.     

The primary structure of the hemoglobin of the Rock-Hopper penguin (Eudyptes crestatus, Sphenisciformes)

The blood of the Rock-Hopper Penguin contains only one hemoglobin component, corresponding to the Hb A of other birds. The primary structures of the alpha- and beta-chains are presented. The chains were separated by high-performance liquid chromatography and cleaved either enzymatically (alpha) or both enzymatically and chemically (beta). Both the native chains and their peptides were sequenced using liquid and gas phase sequenators. The peptides were aligned using their homology to the sequence of human hemoglobin and other bird hemoglobins. As compared to human hemoglobin, 44 amino-acid replacements are found in the alpha-chains (68% homology) and 47 in the beta-chains (67.8% homology). These exchanges involve seven alpha 1/beta 1 and one alpha 1/beta 2 contact in the alpha-chains, whereas in the beta-chains eight alpha 1/beta 1, one alpha 1/beta 2 and one hem contact are substituted. The influence of these replacements on the structure-function relationships in hemoglobin, as well as their importance for the diving ability of penguins, are discussed.     

Reassembly of Lumbricus terrestris hemoglobin: a study by matrix-assisted laser desorption/ionization mass spectrometry and 3D reconstruction from frozen-hydrated specimens

Dodecamers and four types of linker chains (L1-L4) were purified from dissociated hemoglobin of the earthworm Lumbricus terrestris. Various preparations comprising dodecamer of globin chains and linker chains were allowed to reassemble at neutral pH. They produced various oligomers that were purified by gel filtration, analyzed in matrix-assisted laser desorption/ionization mass spectrometry and submitted to 3D reconstruction from isolated particles observed in cryoelectron microscopy. Despite the impossibility to completely free the L2, L3, and L4 preparations from L1, the following conclusions were obtained. First, hemoglobin molecules indistinguishable from native hemoglobin at 25 A resolution were obtained in the absence of linker chains L2, L3, or L4. Second, the 3D reconstruction volumes of reassembled hemoglobins containing dodecamers and L1+L3 or dodecamers and L1+L4 demonstrate that reassembly of native-like structures can be obtained from at most two linker chains and dodecamers. Third, the 3D reconstruction volumes of native and reassembled hemoglobins containing dodecamers and (1) L1, L2, and L4, (2) L1, L3, and L4, (3) L1 and L4, and (4) L1 and L3 were highly similar. Since these structures comprise two types of substructures (one involved in the c3a, c3b, and c4 linking units of the hollow globular substructure and the other in the c5 connection and the toroid), it seems highly probable that the minimal number of linker chains required to reassemble native-like hemoglobin is at most two.     

Peptide end sequencing by orthogonal MALDI tandem mass spectrometry

Highly sensitive peptide fragmentation and identification in sequence databases is a cornerstone of proteomics. Previously, a two-layered strategy consisting of MALDI peptide mass fingerprinting followed by electrospray tandem mass spectrometry of the unidentified proteins has been successfully employed. Here, we describe a high-sensitivity/high-throughput system based on orthogonal MALDI tandem mass spectrometry (o-MALDI) and the automated recognition of fragments corresponding to the N- and C-terminal amino acid residues. Robotic deposition of samples onto hydrophobic anchor substrates is employed, and peptide spectra are acquired automatically. The pulsing feature of the QSTAR o-MALDI mass spectrometer enhances the low mass region of the spectra by approximately 1 order of magnitude. Software has been developed to automatically recognize characteristic features in the low mass region (such as the y1 ion of tryptic peptides), maintaining high mass accuracy even with very low count events. Typically, the sum of the N-terminal two ions (b2 ion), the third N-terminal ion (b3 ion), and the two C-terminal fragments of the peptide (y1 and y2) can be determined. Given mass accuracy in the low ppm range, peptide end sequencing on one or two tryptic peptides is sufficient to uniquely identify a protein from gel samples in the low silver-stained range.     

Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract

Background

Gluten proteins, prominent constituents of barley, wheat and rye, cause celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and the protease-resistant domains contain multiple immunogenic epitopes. The aim of this study was to identify novel sources of gluten-digesting microbial enzymes from the upper gastro-intestinal tract with the potential to neutralize gluten epitopes.

Methodology/Principal Findings

Oral microorganisms with gluten-degrading capacity were obtained by a selective plating strategy using gluten agar. Microbial speciations were carried out by 16S rDNA gene sequencing. Enzyme activities were assessed using gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer α-gliadin and 26-mer γ-gliadin immunogenic peptides. Fragments of the gliadin peptides were separated by RP-HPLC and structurally characterized by mass spectrometry. Strains with high activity towards gluten were typed as Rothia mucilaginosa and Rothia aeria. Gliadins (250 µg/ml) added to Rothia cell suspensions (OD₆₂₀ 1.2) were degraded by 50% after ∼30 min of incubation. Importantly, the 33-mer and 26-mer immunogenic peptides were also cleaved, primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were ∼70–75 kDa in size, and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3–10).

Conclusion/Significance

While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. The identified bacteria may be exploited for physiologic degradation of harmful gluten peptides.     

Location of 3-Hydroxyproline Residues in Collagen Types I, II, III, and V/XI Implies a Role in Fibril Supramolecular Assembly

Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro⁹⁸⁶ in A-clade chains α1(I), α1(II), and α2(V). Two partially modified sites (A2 and A3) were found at Pro⁹⁴⁴ in α1(II) and α2(V) and Pro⁷⁰⁷ in α2(I) and α2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro⁴⁷⁰ in α2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian α1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.     

Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has been applied primarily to the analysis of glycosphingolipids separated from other complex mixtures by TLC, but it is difficult to obtain quantitative profiling of each glycosphingolipid among the different spots on TLC by MALDI-MS. Thus, the development of a convenient approach that utilizes liquid chromatography/electrospray ionization (LC/ESI)-MS has received interest. However, previously reported methods have been insufficient to separate and distinguish each ganglioside class. Here we report an effective method for the targeted analysis of theoretically expected ganglioside molecular species by LC/ESI tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). MRM detection specific for sialic acid enabled us to analyze ganglioside standards such as GM1, GM2, GM3, GD1, and GT1 at picomolar to femtomolar levels. Furthermore, other gangliosides, such as GD2, GD3, GT2, GT3, and GQ1, were also detected in glycosphingolipid standard mixtures from porcine brain and acidic glycolipid extract from mouse brain by theoretically expanded MRM. We found that this approach was also applicable to sulfatides contained in the glycosphingolipid mixtures. In addition, we established a method to separate and distinguish regioisomeric gangliosides, such as GM1a and -1b, GD1a, -1b, and -1c, and GT1a, -1b, and -1c with diagnostic sugar chains in the MRM.     

Biosynthesis of Hb in individual fetal liver bursts : γ-Chain production peaks earlier than β-chain in the erythropoietic pathway

The relative synthesis of α-, β-, ^Gγ- and ^Aγ-globin chains has been evaluated in single fetal liver bursts, which were grown in methylcellulose cultures, individually labelled with [³H]leucine and then analysed via iso-electric focusing. Well-hemoglobinized bursts demonstrate a homogeneous globin synthetic pattern, characterized by prevalent HbF (+some HbA) synthesis: thus, they apparently originate from a homogeneously programmed population of erythroid burst-forming unit (BFU-E). On day 8–9 of culture, the synthetic pattern in ‘mature’ (i.e., well-hemoglobinized) bursts has been compared with that in simultaneously-grown, ‘immature’ (i.e., poorly-hemoglobinized) colonies. These patterns have been further compared with that in ‘matured’ bursts (identified in situ as immature on day 8–9 and labelled 2–4 days later when matured). The ‘immature’ colonies showed very low levels of relative β-globin synthesis, while the ‘mature’ ones demonstrated a more elevated production of β-chain. Significantly, the ‘matured’ bursts showed a globin chain synthetic pattern similar to that of previously labelled ‘matured’ colonies. It is postulated therefore that in fetal liver (and also in adult marrow) the synthesis of γ-chain is linked to an early differentiation stage of erythroblasts, while β-globin synthesis is largely activated at a more advanced maturation stage.     

A comparison of MS/MS‐based,stable‐isotope‐labeled,quantitation performance on ESI‐quadrupole TOF and MALDI‐TOF/TOF mass spectrometers

The peptide‐based quantitation accuracy and precision of LC‐ESI (QSTAR Elite) and LC‐MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ‐labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC‐MALDI spectra. The average protein sequence coverages for LC‐ESI and LC‐MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ‐based expression ratios determined by ProteinPilot from the 57 467 ESI‐MS/MS and 26 085 MALDI‐MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7–6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC‐ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC‐MALDI iTRAQ ratios were rejected. Re‐analysis of an archived LC‐MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS‐based peptide quantitation performance of offline LC‐MALDI was comparable with on‐line LC‐ESI, which required threefold less time. However, offline LC‐MALDI allows the re‐analysis of archived HPLC‐separated samples.     

Bovine hemoglobin: an attractive source of antibacterial peptides

A peptic hemoglobin hydrolysate was fractioned by a semi-preparative reversed-phase HPLC and some fractions have an antibacterial activity against four bacteria strains: Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis. These fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize the peptides in these fractions. Each fraction contains at least three peptides and some fractions contain five peptides. All these fractions were purified several times by HPLC to obtain pure peptides. Thirty antibacterial peptides were identified. From the isolated antibacterial peptides, 24 peptides were derived from the chains of hemoglobin and 6 peptides were derived from the β chains of hemoglobin. The lowest concentration of these peptides (minimum inhibitory concentration (MIC)) necessary to completely inhibit the growth of four bacteria strain was determined. The cell population of all of the tested bacteria species decreased by at least 97% after a 24-h incubation with any of the peptides at the minimum inhibitory concentration.     

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.     

Effect of 2MEGA labeling on membrane proteome analysis using LC-ESI QTOF MS

One of the challenges associated with large-scale proteome analysis using tandem mass spectrometry (MS/MS) and automated database searching is to reduce the number of false positive identifications without sacrificing the number of true positives found. In this work, a systematic investigation of the effect of 2MEGA labeling (N-terminal dimethylation after lysine guanidination) on the proteome analysis of a membrane fraction of an Escherichia coli cell extract by 2-dimensional liquid chromatography MS/MS is presented. By a large-scale comparison of MS/MS spectra of native peptides with those from the 2MEGA-labeled peptides, the labeled peptides were found to undergo facile fragmentation with enhanced a1 or a1-related (a(1)-17 and a(1)-45) ions derived from all N-terminal amino acids in the MS/MS spectra; these ions are usually difficult to detect in the MS/MS spectra of nonderivatized peptides. The 2MEGA labeling alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI and ESI MS experiments. 2MEGA labeling was found not only to increase the number of peptides and proteins identified but also to generate enhanced a1 or a1-related ions as a constraint to reduce the number of false positive identifications. In total, 640 proteins were identified from the E. coli membrane fraction, with each protein identified based on peptide mass and sequence match of one or more peptides using MASCOT database search algorithm from the MS/MS spectra generated by a quadrupole time-of-flight mass spectrometer. Among them, the subcellular locations of 336 proteins are presently known, including 258 membrane and membrane-associated proteins (76.8%). Among the classified proteins, there was a dramatic increase in the total number of integral membrane proteins identified in the 2MEGA-labeled sample (153 proteins) versus the unlabeled sample (77 proteins).     

Reproducibility of an HPLC-ESI-MS/MS method for the measurement of stable-isotope enrichment of in vivo-labeled muscle ATP synthase beta subunit

We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R² = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase_134-143 peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase_134-143 peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R² = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase_134-143 peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase_134-143 peptide.     

A comparison of the tryptic peptides of hemoglobin from two microtine genera: Clethrionomys and Ondatra

The composition of tryptic peptides was determined for Hb (major or fast electrophoretic component) from Clethrionomys rutilus and from Ondatra zibethicus. Analysis of the separated and β-Hb chains made possible the definition of theT9 and βT12 peptides and the resolution of uncertainties in earlier Microtus data for peptides T9,T12b,βT9,βT12 and βT13. The previous postulate of two deletions in peptide βT5 is supported by the present data, and the independent dipeptide βT6 appears in the Ondatra Hb. A Microtus stem sequence is inferred and used for comparison with the new data, and cladograms are given to accomodate the extended phylogenetic progression.