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1.  Quantitative cross-linking/mass spectrometry using isotope-labelled cross-linkers☆ 
Journal of Proteomics  2013;88(100):120-128.
Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue-resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. We found two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible.
Biological significance
Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue-resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so. We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Our work lays the foundations of studying the molecular details of biological processes at greater ease than this could be done so far.
This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].
Graphical abstract
•Quantitative cross-linking using isotope labelling is technically feasible•Commercial cross-linker BS3-d0/4 is suitable for quantitative cross-linking•XiQ, an open source script to extract quantitative data from raw files•Cross-link reference data set to test identification and quantitation software•Isotope labelling reduced the number of identified cross-links
PMCID: PMC3714596  PMID: 23541715
Quantitation; Cross-linking; Structural biology; Protein dynamics; Mass spectrometry; Proteomics
2.  Characterization of the Recombinant Exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 Important for Food Protein Hydrolysis 
PLoS ONE  2013;8(7):e70055.
The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC and the general aminopeptidase N (PepN; EC from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkatH-Ala-Pro-pNA L−1, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkatH-Ala-pNA L−1. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn2+-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co2+>Zn2+>Mn2+>Ca2+>Mg2+. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.
PMCID: PMC3716637  PMID: 23894590
3.  Biotransformation of caffeoyl quinic acids from green coffee extracts by Lactobacillus johnsonii NCC 533 
AMB Express  2013;3:28.
The potential of Lactobacillus johnsonii NCC 533 to metabolize chlorogenic acids from green coffee extract was investigated. Two enzymes, an esterase and a hydroxycinnamate decarboxylase (HCD), were involved in this biotransformation. The complete hydrolysis of 5-caffeoylquinic acid (5-CQA) into caffeic acid (CA) by L. johnsonii esterase occurred during the first 16 h of reaction time. No dihydrocaffeic acid was identified in the reaction mixture. The decarboxylation of CA into 4-vinylcatechol (4-VC) started only when the maximum concentration of CA was reached (10 μmol/ml). CA was completely transformed into 4-VC after 48 h of incubation. No 4-vinylphenol or other derivatives could be identified in the reaction media. In this study we demonstrate the capability of L. johnsonii to transform chlorogenic acids from green coffee extract into 4-VC in two steps one pot reaction. Thus, the enzymatic potential of certain lactobacilli might be explored to generate flavor compounds from plant polyphenols.
PMCID: PMC3679781  PMID: 23692950
Chlorogenic acid; 4-vinylcatechol; Esterase; Decarboxylase; L. johnsonii
4.  Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481 
AMB Express  2012;2:39.
The aminopeptidase P (PepP, EC gene from Lactococcus lactis ssp. lactis DSM 20481 was cloned, sequenced and expressed recombinantly in E. coli BL21 (DE3) for the first time. PepP is involved in the hydrolysis of proline-rich proteins and, thus, is important for the debittering of protein hydrolysates. For accurate determination of PepP activity, a novel gas chromatographic assay was established. The release of L-leucine during the hydrolysis of L-leucine-L-proline-L-proline (LPP) was examined for determination of PepP activity. Sufficient recombinant PepP production was achieved via bioreactor cultivation at 16 °C, resulting in PepP activity of 90 μkatLPP Lculture-1. After automated chromatographic purification by His-tag affinity chromatography followed by desalting, PepP activity of 73.8 μkatLPP Lculture-1 was achieved. This was approximately 700-fold higher compared to the purified native PepP produced by Lactococcus lactis ssp. lactis NCDO 763 as described in literature. The molecular weight of PepP was estimated to be ~ 40 kDa via native-PAGE together with a newly developed activity staining method and by SDS-PAGE. Furthermore, the kinetic parameters Km and Vmax were determined for PepP using three different tripeptide substrates. The purified enzyme showed a pH optimum between 7.0 and 7.5, was most active between 50°C and 60°C and exhibited reasonable stability at 0°C, 20°C and 37°C over 15 days. PepP activity could be increased 6-fold using 8.92 mM MnCl2 and was inhibited by 1,10-phenanthroline and EDTA.
PMCID: PMC3418211  PMID: 22853547
Lactococcus lactis; Aminopeptidase P; PepP; Gas chromatographic assay; Activity staining; LPP
5.  The use of concentrated heat after insect bites/stings as an alternative to reduce swelling, pain, and pruritus: an open cohort-study at German beaches and bathing-lakes 
Swelling, pain, and pruritus are the most relevant symptoms after insect bites/stings. Glucocorticoids and antihistamines are well established in insect sting treatment. Bite Away® is a CE-certified medical device of class 2A (noninvasive device intended for administration to the body, which exchanges energy with the patient in a therapeutic manner) to reduce swelling, pruritus, and pain after insect bites/stings via non-invasive administration of concentrated heat to the skin. We therefore performed a prospective, non-interventional, single-arm cohort study with 146 volunteers using the visual analog scale (VAS) for insect bites/stings to study the reduction of swelling, pruritus, and pain. Demographic data, time from insect sting to treatment, number and duration of administrations of concentrated heat, relevant symptoms, and the development of a VAS score of swelling, pruritus, and pain on baseline, after 2, 5, and 10 minutes after administration, were registered.
In total 146 subjects with a mean age of 29.4 ± 20.7 years (range 2–81) were enrolled in the study. Ninety-three (63.7%) of the subjects were stung by wasps, 33 (22.6%) of the subjects were bitten by mosquitoes, and eight suffered bee stings (5.3%). VAS score swelling decreased with statistical significance after the use of Bite Away® from 4 before treatment to 2 and 1 after 2–5 and 10 minutes, respectively. VAS pain score was 6 before treatment, 2 after 2 minutes, 1 after 5 minutes, and 0 after 10 minutes (median). VAS pruritus score was only available for 52 (35.2%) of the patients. The score decreased from 5 before treatment, to 2 after 2 minutes, and 0 after 5 and 10 minutes.
Locally administrated concentrated heat leads to fast amelioration of symptoms. Usually an absence of symptoms is noticeable 10 minutes after administration. Pain reduction is the dominant effect. Compared with alternatives of pruritus and pain treatment after insect bites/stings, Bite Away® seems to be the fastest treatment option available.
PMCID: PMC3257884  PMID: 22253544
insect bites/stings; swelling; pain; pruritus; concentrated heat; VAS
6.  The Protein Composition of Mitotic Chromosomes Determined Using Multiclassifier Combinatorial Proteomics 
Cell  2010;142(5):810-821.
Despite many decades of study, mitotic chromosome structure and composition remain poorly characterized. Here, we have integrated quantitative proteomics with bioinformatic analysis to generate a series of independent classifiers that describe the ∼4,000 proteins identified in isolated mitotic chromosomes. Integrating these classifiers by machine learning uncovers functional relationships between protein complexes in the context of intact chromosomes and reveals which of the ∼560 uncharacterized proteins identified here merits further study. Indeed, of 34 GFP-tagged predicted chromosomal proteins, 30 were chromosomal, including 13 with centromere-association. Of 16 GFP-tagged predicted nonchromosomal proteins, 14 were confirmed to be nonchromosomal. An unbiased analysis of the whole chromosome proteome from genetic knockouts of kinetochore protein Ska3/Rama1 revealed that the APC/C and RanBP2/RanGAP1 complexes depend on the Ska complex for stable association with chromosomes. Our integrated analysis predicts that up to 97 new centromere-associated proteins remain to be discovered in our data set.
Graphical Summary
► Method to define the protein composition of a complex nonpurifiable organelle ► Combines genetics and proteomics to study complexes in whole chromosomes ► Use of machine learning to uncover functional relationships between proteins ► Comprehensive list of >4000 mitotic chromosome-associated proteins
PMCID: PMC2982257  PMID: 20813266
7.  Architecture of the RNA polymerase II–TFIIF complex revealed by cross-linking and mass spectrometry 
The EMBO Journal  2010;29(4):717-726.
Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes.
PMCID: PMC2810376  PMID: 20094031
higher-order protein complex; integrated structure analysis; mass spectrometry; multi-dimensional structure and dynamics of biological macromolecules; transcription and its regulation
8.  Production of a New d-Amino Acid Oxidase from the Fungus Fusarium oxysporum 
The fungus Fusarium oxysporum produced a d-amino acid oxidase (EC in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific d-amino acid oxidase activity was increased up to 12.5-fold with various d-amino acids or their corresponding derivatives as inducers. The best inducers were d-alanine (2.7 μkat/g of dry biomass) and d-3-aminobutyric acid (2.6 μkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal d-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric d-amino acid oxidase activity of 17 μkat liter−1 with d-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 μkat liter−1 (3,240 U liter−1).
PMCID: PMC91565  PMID: 10427080

Results 1-8 (8)