A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed

Olsen, Jesper V.; Schwartz, Jae C.; Griep-Raming, Jens; Nielsen, Michael L.; Damoc, Eugen; Denisov, Eduard; Lange, Oliver; Remes, Philip M.; Taylor, Dennis K.; Splendore, Maurizio; Wouters, Eloy R.; Senko, Michael W.; Makarov, Alexander; Mann, Matthias; Horning, Stevan

doi:10.1074/mcp.m900375-mcp200

Cited by 422 publications

(450 citation statements)

References 20 publications

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“…The eluate was sprayed directly into a mass spectrometer (LTQ-Orbitrap-XL-ETD; Thermo Fisher Sci- entific) for analysis (19). The repetitive analytical cycle comprised two stages.…”

Section: Methodsmentioning

confidence: 99%

“…To map the site of covalent guanylylation within the 408-aa RtcB polypeptide, we reacted the recombinant protein in vitro with unlabeled GTP, then subjected the sample to cysteine reduction and alkylation with iodoacetamide, followed by proteolysis with trypsin. The tryptic peptides were analyzed by HPLC interfaced with nano-electrospray ionization mass spectrometry (19). We thereby identified a species with a m/z value of 723.93, consistent with a GMP adduct of the tryptic peptide 325 GLGNEESFCSCSHGAGR 341 (measured molecular mass 2,168.777; calculated molecular mass 2,168.778) in which both cysteines were alkylated and the net charge is +3 ( Fig.…”

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confidence: 99%

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RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates

Chakravarty

Subbotin²,

Chait³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

105

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A cherished tenet of nucleic acid enzymology holds that synthesis of polynucleotide 3′-5′ phosphodiesters proceeds via the attack of a 3′-OH on a high-energy 5′ phosphoanhydride: either a nucleoside 5′-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A(5′)pp(5′)N-in the case of polynucleotide ligases. RtcB exemplifies a family of RNA ligases implicated in tRNA splicing and repair. Unlike classic ligases, RtcB seals broken RNAs with 3′-phosphate and 5′-OH ends. Here we show that RtcB executes a three-step ligation pathway entailing (i) reaction of His337 of the enzyme with GTP to form a covalent RtcB-(histidinyl-N)-GMP intermediate; (ii) transfer of guanylate to a polynucleotide 3′-phosphate to form a polynucleotide-(3′)pp(5′)G intermediate; and (iii) attack of a 5′-OH on the -N(3′)pp(5′)G end to form the splice junction. RtcB is structurally sui generis, and its chemical mechanism is unique. The wide distribution of RtcB proteins in bacteria, archaea, and metazoa raises the prospect of an alternative enzymology based on covalently activated 3′ ends. RNA or A(5′)pp(5′)DNA; and (iii) ligase catalyzes attack by a polynucleotide 3′-OH on A(5′)pp(5′)RNA/DNA to form a 3′-5′ phosphodiester bond and release AMP (1). The salient principle of classic ligase catalysis is that the high energy of the ATP phosphoanhydride bond is transferred via the enzyme to the polynucleotide 5′ end, thereby activating the 5′ end for phosphodiester synthesis, with AMP as a favorable leaving group. The shared chemical mechanism of classic RNA and DNA ligases is reflected in their conserved core tertiary structures and active sites (1).Classic ATP-dependent RNA ligases are encoded by diverse taxa in all three phylogenetic domains. In bacteria, fungi, and plants they function as components of multienzyme RNA repair pathways that heal and seal broken RNAs with 2′,3′ cyclic phosphate and 5′-OH ends (2-5). In the healing phase, the 2′,3′ cyclic phosphate end is hydrolyzed by a phosphoesterase enzyme to a 3′-OH, and the 5′-OH end is phosphorylated by a polynucleotide kinase enzyme to yield a 5′-monophosphate. The resulting 3′-OH and 5′-phosphate ends are then suitable for ATP-dependent sealing by RNA ligase. This "healing-and-sealing" pathway is responsible for tRNA splicing in fungi and plants (6, 7), for mRNA splicing in the fungal unfolded protein response (8), and for tRNA restriction repair during bacteriophage infection of Escherichia coli (9).An alternative "direct ligation" pathway for joining 2′,3′ cyclic phosphate and 5′-OH ends during mammalian tRNA splicing was discovered nearly 30 y ago (10-12), when it was shown that the 2′,3′ cyclic phosphate of the cleaved pre-tRNA is incorporated at the splice junction in the mature tRNA. (The junction phosphate in yeast tRNA splicing derives from the γ-phosphate of the NTP substrate for the 5′ kinase step.) Direct ligation languished until 2011, when three laboratories identified bacterial, archaeal, and mammalian RtcB proteins as RNA ligase enzymes capable of seali...

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“…The eluate was sprayed directly into a mass spectrometer (LTQ-Orbitrap-XL-ETD; Thermo Fisher Sci- entific) for analysis (19). The repetitive analytical cycle comprised two stages.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates

Chakravarty

Subbotin²,

Chait³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

105

View full text Add to dashboard Cite

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“…The enabling technology was an ETD-enabled LTQ Orbitrap Velos hybrid mass spectrometer exhibiting low ppm mass accuracy, high resolution MS, and MS n capabilities combined with multiple options for performing peptide dissociation (47). We began by using multiple proteases to generate a diverse pool of peptides amenable to MS n (45).…”

Section: A High Mass Accuracy High Resolution Tandem Mass Spectrometmentioning

confidence: 99%

“…An estimated 1-4 g of protein digest was loaded onto the precolumn and chromatographed over a 60-min linear gradient at 300 nl/min (2 to 30% B, buffer A (0.2% formic acid in water); buffer B (0.2% formic acid in acetonitrile)). Eluate was introduced into the mass spectrometer via electrospray ionization (ϩ2.0 kV), and peptide cations were subjected to tandem mass spectrometry using an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) enabled for ETD (39,47,48). Typical experiments consisted of MS 1 analysis in the Orbitrap mass analyzer using a resolving power of 60,000 followed by 10 data-dependent HCD MS 2 events.…”

Section: Liquid Chromatography Electrospray Tandem Mass Spectrometry mentioning

confidence: 99%

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

Yang

Park

Davis

et al. 2012

Journal of Biological Chemistry

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Background: ␣1(V) is an extensively modified collagen chain important in disease. Results: Comprehensive mapping of ␣1(V) post-translational modifications reveals unexpectedly large numbers of X-position hydroxyprolines in Gly-X-Y amino acid triplets. Conclusion:The unexpected abundance of X-position hydroxyprolines suggests a mechanism for differential modification of collagen properties. Significance: Positions, numbers, and occupancy of modified sites can provide insights into ␣1(V) biological properties.

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“…Especially the recently developed Orbitrap Velos shows great promise for indepth and accurate proteome analysis by combining a fast and sensitive dual-pressure linear ion trap with a high accuracy Orbitrap mass analyzer. In addition, an improved higher energy collision-induced dissociation (HCD) collision cell enables acquisition of MS and MS/MS spectra at a very high mass accuracy 41 .…”

Section: Lc-ms Measurementmentioning

confidence: 99%

Toward Quantitative Proteomics of Organ Substructures: Implications for Renal Physiology

Velić

Maček

Wagner

2010

Seminars in Nephrology

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AcknowledgementsResearch in the laboratories of the authors has been supported by grants from the Minstry of Science, Baden-Wuerttenberg (to BM) and the Swiss National Science Foundation, the 6 th and 7 th EU Framework projects (EuReGene and EUNEFRON) (to CAW). We thank MatthiasMann for critically reading and discussing the manuscript. Summary 2Organs are complex structures that consist of multiple tissues with different levels of gene expression. To achieve a comprehensive coverage and accurate quantitation data, organs should ideally be separated into morphological and/or functional substructures prior to gene or protein expression analysis. However, due to complex morphology and elaborate isolation protocols, this was so far often difficult to achieve. Kidneys are organs where functional and morphological subdivision is especially important. Each subunit of the kidney, the nephron, itself consists of more than 10 subsegments with distinct morphological and functional characteristics. For a full understanding of kidney physiology, global gene and protein expression analyses have to be performed at the level of the nephron subsegments; however, such studies have so far been extremely rare.Here we describe the latest approaches in quantitative high accuracy mass spectrometrybased proteomics and their application to quantitative proteomics studies of the whole kidney and nephron subsegments, both in humans and in animal models. We compare these studies with similar studies performed on other organ substructures. We argue that the newest technologies used for preparation, processing and measurement of small amounts of starting material are finally enabling global and subsegment-specific quantitative measurement of protein levels in the kidney and other organs. These new technologies and approaches are making a decisive impact on our understanding of the (patho)physiological processes at the molecular level.3

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A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed

Cited by 422 publications

References 20 publications

RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates

RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

Toward Quantitative Proteomics of Organ Substructures: Implications for Renal Physiology

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