Nicolai Bache

Thermo Fisher Scientific, Waltham, Massachusetts, United States

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Publications (17)117.1 Total impact

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    ABSTRACT: The high peptide sequencing speed provided by modern hybrid tandem mass spectrometers enable the utilisation of fast liquid chromatographic (LC) separation techniques. We present a robust solid phase extraction/capillary LC system (speLC) for 5-10 minute separation of semi-complex peptide mixtures prior to ESI MS/MS for peptide sequencing. This speLC-MS/MS system eliminates sample to sample carry-over by using disposable micropipette solid phase extraction tips (StageTips) for peptide sample loading, concentration and desalting. Automated analysis of 192 replicates of E. coli peptide mixtures in 30 hours demonstrated the throughput, stability and reproducibility of the system. The speLC-MS/MS system detected low-femtomole amounts of peptides and allowed sequencing of 1 microgram of HeLa cells protein extracts at a rate of approximately 90 peptides/minute, identifying more than 1500 peptides (>500 proteins) in a 10-min speLC-MS/MS experiment. Analysis by selected reaction monitoring by speLC-SRM-MS/MS of distinct peptides derived from the blood proteins IGF1, IGF2, IBP2 and IBP3 demonstrated protein quantification with CV values below 10% across 96 replicates. The speLC-MS/MS system is ideally suited for fast screening and characterisation of large numbers of peptide-containing samples in biological, biomedical and clinical laboratories.
    Journal of Proteome Research 10/2014; · 5.06 Impact Factor
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    ABSTRACT: Biomarker analysis of blood samples by liquid chromatography (LC) mass spectrometry (MS) is extremely challenging due to the high protein concentration range, characterised by abundant proteins that suppress and mask other proteins of lower abundance. This situation is further aggravated when using fast high-throughput methods, which are necessary for analysis of hundreds and thousands of samples in clinical laboratories. The blood proteins IGF1, IGF2, IBP2, IBP3 and A2GL have been proposed as indirect biomarkers for detection of GH administration and as putative biomarkers for breast cancer diagnosis. We describe a sensitive and scalable method to quantify these 5 proteins of medium and low abundance by Selected Reaction Monitoring (SRM) LC-MS/MS analysis in blood samples. Our method requires 7 μL of plasma and reaches a throughput of up to ca. 80 analyses per day. It includes an initial protein precipitation protocol optimised for extraction of low mass proteins from blood samples for reduced signal suppression and increased sensitivity in LC-MS/MS. We benchmarked this method for the analysis of 40 individual blood samples including 20 patients diagnosed with breast cancer. Biological significance The interest for MS-based biomarker analysis in body fluids is steadily increasing as proteomics methodology translates into clinical laboratories. We describe a method for detection of 5 distinct proteins of low mass and medium to low abundance, which are of interest in anti-doping and clinical analysis. The analytical setup is simple and robust and is suitable for high-throughput instrument configurations.
    Journal of Proteomics. 09/2014;
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    ABSTRACT: Mass spectrometry has become a powerful tool for measuring protein hydrogen exchange and thereby reveal the structural dynamics of proteins in solution. Here we describe the successful application of a matrix-assisted laser desorption ionization (MALDI) mass spectrometry approach based on in-source decay (ISD) to measure spatially resolved amide backbone hydrogen exchange. By irradiating deuterated protein molecules in a crystalline matrix with a high laser fluence, they undergo prompt fragmentation. Spatially resolved deuteration levels are readily obtained by mass analysis of consecutive fragment ions. MALDI ISD analysis of deuterated cytochrome c yielded an extensive series of c-fragment ions which originate from cleavage of nearly all N-C(α) bonds (Cys17 to Glu104) allowing for a detailed analysis of the deuterium content of the backbone amides. While hydrogen scrambling can be major concern when using mass spectrometric fragmentation to obtain detailed information on protein hydrogen exchange, we show that the level of hydrogen scrambling in our MALDI ISD measurements is negligible and that the known dynamic behavior of cytochrome c in solution is accurately reflected in the deuterium contents of the fragment ions. The developed method combines several attractive features from a practical point of view as it is simple to perform and it readily provides a detailed mapping of the dynamic structure of a protein in solution.
    Analytical Chemistry 11/2011; 83(23):8859-62. · 5.82 Impact Factor
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    ABSTRACT: Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved in the assembly of clathrin coated vesicles in synaptic vesicle endocytosis. Unlike other types of O-glycosylation, O-GlcNAc is nucleocytoplasmic and reversible. It was thought to be a terminal modification, that is, the O-GlcNAc was not found to be additionally modified in any way. We now show that AP180 purified from rat brain contains a phosphorylated O-GlcNAc (O-GlcNAc-P) within a highly conserved sequence. O-GlcNAc or O-GlcNAc-P, but not phosphorylation alone, was found at Thr-310. Analysis of synthetic GlcNAc-6-P produced identical fragmentation products to GlcNAc-P from AP180. Direct O-linkage of GlcNAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification.
    Journal of Proteome Research 06/2011; 10(6):2725-33. · 5.06 Impact Factor
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    ABSTRACT: Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.
    Journal of the American Society for Mass Spectrometry 06/2011; 22(6):989-96. · 3.59 Impact Factor
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    ABSTRACT: Glycogen synthase kinase 3 (GSK3) is a critical enzyme in neuronal physiology; however, it is not yet known whether it has any specific role in presynaptic function. We found that GSK3 phosphorylates a residue on the large GTPase dynamin I (Ser-774) both in vitro and in primary rat neuronal cultures. This was dependent on prior phosphorylation of Ser-778 by cyclin-dependent kinase 5. Using both acute inhibition with pharmacological antagonists and silencing of expression with short hairpin RNA, we found that GSK3 was specifically required for activity-dependent bulk endocytosis (ADBE) but not clathrin-mediated endocytosis. Moreover we found that the specific phosphorylation of Ser-774 on dynamin I by GSK3 was both necessary and sufficient for ADBE. These results demonstrate a presynaptic role for GSK3 and they indicate that a protein kinase signaling cascade prepares synaptic vesicles for retrieval during elevated neuronal activity.
    Nature Neuroscience 07/2010; 13(7):845-51. · 15.25 Impact Factor
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    ABSTRACT: In this study Atlantic cod (Gadus morhua) were exposed to different levels of North Sea produced water (PW) and 17beta-oestradiol (E(2)), a natural oestrogen, from egg to fry stage (90 days). By comparing changes in protein expression following E(2) exposure to changes induced by PW treatment, we were able to compare the induced changes by PW to the mode of action of oestrogens. Changes in the proteome in response to exposure in whole cod fry (approximately 80 days post-hatching, dph) were detected by two-dimensional gel electrophoresis and image analysis and identified by MALDI-ToF-ToF mass spectrometry, using a newly developed cod EST database and the NCBI database. Many of the protein changes occurred at low levels (0.01% and 0.1% PW) of exposure, indicating putative biological responses at lower levels than previously detected. Using discriminant analysis, we identified a set of protein changes that may be useful as biomarker candidates of produced water (PW) and oestradiol exposure in Atlantic cod fry. The biomarker candidates discovered in this study may, following validation, prove effective as diagnostic tools in monitoring exposure and effects of discharges from the petroleum industry offshore, aiding future environmental risk analysis and risk management.
    Aquatic toxicology (Amsterdam, Netherlands) 03/2010; 96(4):280-9. · 3.12 Impact Factor
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    ABSTRACT: The photoneuroendocrine circadian system of the brain consists of (a) specialized photoreceptors in the retina, (b) a circadian generator located in the forebrain that contains "clock genes," (c) specialized nuclei in the forebrain involved in neuroendocrine secretion, and (d) the pineal gland. The circadian generator is a nucleus, called the suprachiasmatic nucleus (SCN). The neurons of this nucleus contain "clock genes," the transcription of which exhibits a circadian rhythm. Most circadian rhythms are generated by the neurons of this nucleus and, via neuronal and humoral connections, the SCN controls circadian activity of the brain and peripheral tissues. The endogenous oscillator of the SCN is each day entrained to the length of the daily photoperiod by light that reach the retina, and specialized photoreceptors transmit impulses to the SCN via the optic nerves. Mass screening for day/night variations in gene expression in the circadian system as well as in the whole brain and peripheral tissues have, during the last decade, been performed. However, studies of circadian changes in the proteome have been less investigated. In this survey, the anatomy and function of the circadian-generating system in mammals is described, and recent proteomic studies that investigate day/night changes in the retina, SCN, and pineal gland are reviewed. Further circadian changes controlled by the SCN in gene and protein expression in the liver are discussed.
    Mass Spectrometry Reviews 06/2009; 29(2):313-25. · 7.74 Impact Factor
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    ABSTRACT: We have previously shown that peptide amide hydrogens undergo extensive intramolecular migration (i.e., complete hydrogen scrambling) upon collisional activation of protonated peptides (Jørgensen et al. J. Am. Chem. Soc. 2005, 127, 2785-2793). The occurrence of hydrogen scrambling enforces severe limitations on the application of gas-phase fragmentation as a convenient method to obtain information about the site-specific deuterium uptake for proteins and peptides in solution. To investigate whether deprotonated peptides exhibit a lower level of scrambling relative to their protonated counterparts, we have now measured the level of hydrogen scrambling in a deprotonated, selectively labeled peptide using MALDI tandem time-of-flight mass spectrometry. Our results conclusively show that hydrogen scrambling is prevalent in the deprotonated peptide upon collisional activation. The amide hydrogens ((1)H/(2)H) have migrated extensively in the anionic peptide, thereby erasing the original regioselective deuteration pattern obtained in solution.
    Journal of the American Society for Mass Spectrometry 12/2008; 19(12):1719-25. · 3.59 Impact Factor
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    ABSTRACT: In mammals, Cytochrome P450 (CYP) enzymes are bound to membranes of the endoplasmic reticulum and mitochondria, where they are responsible for the oxidative metabolism of many xenobiotics as well as organic endogenous compounds. In humans, 57 isoforms were identified which are classified based on sequence homology. In the present work, we demonstrate the performance of a mass spectrometry-based strategy to simultaneously detect and differentiate distinct human Cytochrome P450 (CYP) isoforms including the highly similar CYP3A4, CYP3A5, CYP3A7, as well as CYP2C8, CYP2C9, CYP2C18, CYP2C19, and CYP4F2, CYP4F3, CYP4F11, CYP4F12. Compared to commonly used immunodetection methods, mass spectrometry overcomes limitations such as low antibody specificity and offers high multiplexing possibilities. Furthermore, CYP phosphorylation, which may affect various biochemical and enzymatic properties of these enzymes, is still poorly analyzed, especially in human tissues. Using titanium dioxide resin combined with tandem mass spectrometry for phosphopeptide enrichment and sequencing, we discovered eight human P450 phosphorylation sites, seven of which were novel. The data from surgical human liver samples establish that the isoforms CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2D6, CYP3A4, CYP3A7, and CYP8B1 are phosphorylated in vivo. These results will aid in further investigation of the functional significance of protein phosphorylation for this important group of enzymes.
    Journal of Proteome Research 11/2008; 7(11):4678-88. · 5.06 Impact Factor
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    ABSTRACT: To achieve a fundamental understanding of the function of proteins and protein complexes at the molecular level, it is crucial to obtain a detailed knowledge about their dynamic and structural properties. The kinetics of backbone amide hydrogen exchange is intimately linked to the structural dynamics of the protein, and in recent years, the monitoring of the isotopic exchange of these hydrogens by mass spectrometry has become a recognized method. At present, the resolution of this method is, however, limited and single-residue resolution is typically only obtained for a few residues in a protein. It would therefore be desirable if gas-phase fragmentation could be used to localize incorporated deuterons as this would ultimately lead to single-residue resolution. A central obstacle for this approach is, however, the occurrence of intramolecular migration of amide hydrogens upon activation of the gaseous protein (i.e., hydrogen scrambling). Here we investigate the occurrence of scrambling in selectively labeled peptides upon fragmentation by matrix-assisted laser desorption/ionization in-source decay (MALDI ISD). We have utilized peptides with a unique regioselective deuterium incorporation that allows us to accurately determine the extent of scrambling upon fragmentation. Our results show that the level of scrambling upon MALDI ISD is so low that the solution deuteration pattern is readily apparent in the gas-phase fragment ions. These results suggest that MALDI ISD may prove useful for hydrogen exchange studies of purified peptides and small proteins.
    Analytical Chemistry 08/2008; 80(16):6431-5. · 5.82 Impact Factor
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    ABSTRACT: Amphiphysin I (amphI) is dephosphorylated by calcineurin during nerve terminal depolarization and synaptic vesicle endocytosis (SVE). Some amphI phosphorylation sites (phosphosites) have been identified with in vitro studies or phosphoproteomics screens. We used a multifaceted strategy including 32P tracking to identify all in vivo amphI phosphosites and determine their relative abundance and potential relevance to SVE. AmphI was extracted from 32P-labeled synaptosomes, phosphopeptides were isolated from proteolytic digests using TiO2 chromatography, and mass spectrometry revealed 13 sites: serines 250, 252, 262, 268, 272, 276, 285, 293, 496, 514, 539, and 626 and Thr-310. These were distributed into two clusters around the proline-rich domain and the C-terminal Src homology 3 domain. Hierarchical phosphorylation of Ser-262 preceded phosphorylation of Ser-268, -272, -276, and -285. Off-line HPLC separation and two-dimensional tryptic mapping of 32P-labeled amphI revealed that Thr-310, Ser-293, Ser-285, Ser-272, Ser-276, and Ser-268 contained the highest 32P incorporation and were the most stimulus-sensitive. Individually Thr-310 and Ser-293 were the most abundant phosphosites, incorporating 16 and 23% of the 32P. The multiple phosphopeptides containing Ser-268, Ser-276, Ser-272, and Ser-285 had 27% of the 32P. Evidence for a role for at least one proline-directed protein kinase and one non-proline-directed kinase was obtained. Four phosphosites predicted for non-proline-directed kinases, Ser-626, -250, -252, and -539, contained low amounts of 32P and were not depolarization-responsive. At least one alternatively spliced amphI isoform was identified in synaptosomes as being constitutively phosphorylated because it did not incorporate 32P during the 1-h labeling period. Multiple phosphosites from amphI-co-migrating synaptosomal proteins were also identified, including SGIP (Src homology 3 domain growth factor receptor-bound 2 (Grb2)-like (endophilin)-interacting protein 1), AAK1, eps15R, MAP6, alpha/beta-adducin, and HCN1. The results reveal two sets of amphI phosphosites that are either dynamically turning over or constitutively phosphorylated in nerve terminals and improve understanding of the role of individual amphI sites or phosphosite clusters in synaptic SVE.
    Molecular &amp Cellular Proteomics 07/2008; 7(6):1146-61. · 7.25 Impact Factor
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    ABSTRACT: The pineal gland secretes the hormone melatonin. This secretion exhibits a circadian rhythm with a zenith during night and a nadir during day. We have performed proteome analysis of the superficial pineal gland in rats during daytime and nighttime. The proteins were extracted and subjected to 2-DE. Of 1747 protein spots revealed by electrophoresis, densitometric analysis showed the up-regulation of 25 proteins during nighttime and of 35 proteins during daytime. Thirty-seven of the proteins were identified by MALDI-TOF MS. The proteins up-regulated during the night are involved in the Krebs cycle, energy transduction, calcium binding, and intracellular transport. During the daytime, enzymes involved in glycolysis, electron transport, and also the Krebs cycle were up-regulated as well as proteins taking part in RNA binding and RNA processing. Our data show a prominent day-night variation of the protein levels in the rat pineal gland. Some proteins are up-regulated during the night concomitant with the melatonin secretion of the gland. Other proteins are up-regulated during the day indicating a pineal metabolism not related to the melatonin synthesis.
    PROTEOMICS 07/2007; 7(12):2009-18. · 4.13 Impact Factor
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    ABSTRACT: Dynamin I (dynI) is phosphorylated in synaptosomes at Ser(774) and Ser(778) by cyclin-dependent kinase 5 to regulate recruitment of syndapin I for synaptic vesicle endocytosis, and in PC12 cells on Ser(857). Hierarchical phosphorylation of Ser(774) precedes phosphorylation of Ser(778). In contrast, Thr(780) phosphorylation by cdk5 has been reported as the sole site (Tomizawa, K., Sunada, S., Lu, Y. F., Oda, Y., Kinuta, M., Ohshima, T., Saito, T., Wei, F. Y., Matsushita, M., Li, S. T., Tsutsui, K., Hisanaga, S. I., Mikoshiba, K., Takei, K., and Matsui, H. (2003) J. Cell Biol. 163, 813-824). To resolve the discrepancy and to better understand the biological roles of dynI phosphorylation, we undertook a systematic identification of all phosphorylation sites in rat brain nerve terminal dynI. Using phosphoamino acid analysis, exclusively phospho-serine residues were found. Thr(780) phosphorylation was not detectable. Mutation of Ser(774), Ser(778), and Thr(780) confirmed that Thr(780) phosphorylation is restricted to in vitro conditions. Mass spectrometry of (32)P-labeled phosphopeptides separated by two-dimensional mapping revealed seven in vivo phosphorylation sites: Ser(774), Ser(778), Ser(822), Ser(851), Ser(857), Ser(512), and Ser(347). Quantification of (32)P radiation in each phosphopeptide showed that Ser(774) and Ser(778) were the major sites (up to 69% of the total), followed by Ser(851) and Ser(857) (12%), and Ser(853) (2%). Phosphorylation of Ser(851) and Ser(857) was restricted to the long tail splice variant dynIxa and was not hierarchical. Co-purified, (32)P-labeled dynIII was phosphorylated at Ser(759), Ser(763), and Ser(853). Ser(853) is homologous to Ser(851) in dynIxa. The results identify all major and several minor phosphorylation sites in dynI and provide the first measure of their relative abundance and relative responses to depolarization. The multiple phospho-sites suggest subtle regulation of synaptic vesicle endocytosis by new protein kinases and new protein-protein interactions. The homologous dynI and dynIII phosphorylation indicates a high mechanistic similarity. The results suggest a unique role for the long splice variants of dynI and dynIII in nerve terminals.
    Journal of Biological Chemistry 06/2007; 282(20):14695-707. · 4.65 Impact Factor
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    ABSTRACT: Telomerase is a ribonucleoprotein enzyme complex that adds 5'-TTAGGG-3' repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for approximately 90% of human cancers. We have purified human telomerase approximately 10(8)-fold, with the final elution dependent on the enzyme's ability to catalyze nucleotide addition onto a DNA oligonucleotide of telomeric sequence, thereby providing specificity for catalytically active telomerase. Mass spectrometric sequencing of the protein components and molecular size determination indicated an enzyme composition of two molecules each of telomerase reverse transcriptase, telomerase RNA, and dyskerin.
    Science 03/2007; 315(5820):1850-3. · 31.20 Impact Factor
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    ABSTRACT: Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.
    Rapid Communications in Mass Spectrometry 02/2006; 20(7):1127-34. · 2.51 Impact Factor
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    ABSTRACT: Considerable controversy exists in the literature as to the occurrence of intramolecular migration of amide hydrogens upon collisional activation of protonated peptides and proteins. This phenomenon has important implications for the application of CID as an experimental tool to obtain site-specific information about the incorporation of deuterium into peptides and proteins in solution. Using a unique set of peptides with their carboxyl-terminal half labeled with deuterium we have shown unambiguously that hydrogen (1H/2H) scrambling is such a dominating factor during low energy collisional activation of doubly protonated peptides that the original regioselective deuterium pattern of these peptides is completely erased (Jørgensen, T. J. D., Gårdsvoll, H., Ploug, M., and Roepstorff, P. (2005) Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation. J. Am. Chem. Soc.127, 2785-2793). Taking further advantage of this unique test system we have now investigated the influence of the charge state and collision energy on the occurrence of scrambling in protonated peptides. Our MALDI tandem time-of-flight experiments clearly demonstrate that complete positional randomization among all exchangeable sites (i.e. all N- and O-linked hydrogens) also occurs upon high energy collisional activation of singly protonated peptides. This intense proton/deuteron traffic precludes the use of MALDI tandem time-of-flight mass spectrometry to obtain reliable information on the specific incorporation pattern of deuterons obtained during exchange experiments in solution.
    Molecular &amp Cellular Proteomics 01/2006; 4(12):1910-9. · 7.25 Impact Factor

Publication Stats

456 Citations
117.10 Total Impact Points

Institutions

  • 2014
    • Thermo Fisher Scientific
      Waltham, Massachusetts, United States
  • 2008–2011
    • University of Sydney
      • Children's Medical Research Institute (CMRI)
      Sydney, New South Wales, Australia
  • 2007–2008
    • University of Southern Denmark
      • Department of Biochemistry and Molecular Biology
      Copenhagen, Capital Region, Denmark
    • Cell Signaling Technology
      Beverly, Massachusetts, United States