Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT

McDonald, W. Hayes; Ohi, Ryoma; Miyamoto, David T.; Mitchison, Timothy J.; Yates, John R.

doi:10.1016/s1387-3806(02)00563-8

Cited by 284 publications

(258 citation statements)

References 19 publications

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“…One such instrument that has recently been commercialized is the LTQ-Orbitrap hybrid mass spectrometer. As an initial evaluation, we examined a collection of unlabeled and metabolically 15 N labeled yeast standards that were mixed in known ratios. These samples provided the basis for a well-controlled set of experiments aimed at probing the quantitative analysis capabilities of the LTQ-Orbitrap.…”

Section: Resultsmentioning

confidence: 99%

“…The measurements from the Orbitrap dataset were generally more accurate and of higher quality compared to the LTQ measurements and convincingly demonstrated the performance advantages of the Orbitrap versus the LTQ for quantitative analyses. To further demonstrate the improved quantitative analysis capabilities of the Orbitrap, we also quantitatively analyzed changes in protein expression in 15 N metabolically labeled ratderived synaptosome samples during the course of rat brain development and successfully identified 149 synaptic proteins whose abundance changes during development. …”

Section: Introductionmentioning

confidence: 99%

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Relative Quantification of Stable Isotope Labeled Peptides Using a Linear Ion Trap-Orbitrap Hybrid Mass Spectrometer

et al. 2007

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The quantitative analysis of complex biological samples has emerged as a key research area in the field of proteomics. Although quantitative proteomic experiments remain challenging, these strategies have been greatly facilitated by the development of newer high-performance mass spectrometers. In this work, we have evaluated the use of the LTQ-Orbitrap, a hybrid mass spectrometer in which a linear ion trap is coupled to an Orbitrap mass analyzer, for quantitative analyses. By analyzing a range of yeast protein standards, we found that the high mass accuracy, high resolution, large ion capacity, and large dynamic range of the LTQ-Orbitrap led to a significant improvement in the number and quality of the peptide ratio measurements compared to similar analyses done on the LTQ. We also successfully quantified protein expression differences that occur in metabolically labeled rat synapses during brain development to further demonstrate the suitability of the LTQ-Orbitrap for the comparative analysis of complex tissue samples.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Relative Quantification of Stable Isotope Labeled Peptides Using a Linear Ion Trap-Orbitrap Hybrid Mass Spectrometer

et al. 2007

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“…Formic acid (5% final) was added to stop the reactions. Samples were loaded on split triple-phase fused silica microcapillary columns (33) and placed in line with an Eksigent NanoLC 2D system. Full MS spectra were recorded on the peptides over a 400 -1700 m/z range in the Orbitrap at 60,000 resolution, followed by fragmentation in the ion trap (at 35% collision energy) on the first to fifth most intense ions selected from the full MS spectrum with dynamic exclusion enabled for 90 s (34).…”

Section: Methodsmentioning

confidence: 99%

Unraveling the Ubiquitome of the Human Malaria Parasite

Ponts

Saraf

Chung

et al. 2011

Journal of Biological Chemistry

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Malaria is one of the deadliest infectious diseases worldwide. The most severe form is caused by the eukaryotic protozoan parasite Plasmodium falciparum. Recent studies have highlighted the importance of post-translational regulations for the parasite's progression throughout its life cycle, protein ubiquitylation being certainly one of the most abundant. The specificity of its components and the wide range of biological processes in which it is involved make the ubiquitylation pathway a promising source of suitable targets for anti-malarial drug development. Here, we combined immunofluorescent microscopy, biochemical assays, in silico prediction, and mass spectrometry analysis using the multidimensional protein identification technology, or MudPIT, to describe the P. falciparum ubiquitome. We found that ubiquitin conjugates are detected at every morphological stage of the parasite erythrocytic cycle. Furthermore, we detected that more than half of the parasite's proteome represents possible targets for ubiquitylation, especially proteins found to be present at the most replicative stage of the asexual cycle, the trophozoite stage. A large proportion of ubiquitin conjugates were also detected at the schizont stage, consistent with a cell activity slowdown to prepare for merozoite differentiation and invasion. Finally, for the first time in the human malaria parasite, our results strongly indicate the presence of heterologous mixed conjugations, SUMO/UB. This discovery suggests that sumoylated proteins may be regulated by ubiquitylation in P. falciparum. Altogether, our results present the first stepping stone toward a better understanding of ubiquitylation and its role(s) in the biology of the human malaria parasite.

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“…The sample was then sequentially digested with endoproteinase Lys-C (Roche Diagnostics) and sequencing grade soluble trypsin (Promega) to produce a large collection of peptide fragments (14). The resulting peptide mixture was then separated and analyzed by multidimensional protein identification technology, which features a series of HPLC columns (reverse phase/strong cation exchanger/reverse phase) followed by electrospray ionization tandem mass spectrometry as described previously (15,19), with modifications as described in McDonald et al (17). Tandem mass spectra were searched against the rat, mouse, and human translated genomic databases using the SEQUEST software algorithm (20,21).…”

Section: Multidimensional High Pressure Liquid Chromatography Coupledmentioning

confidence: 99%

Protein Trafficking and Anchoring Complexes Revealed by Proteomic Analysis of Inward Rectifier Potassium Channel (Kir2.x)-associated Proteins

Leonoudakis

Conti

Anderson

et al. 2004

Journal of Biological Chemistry

196

148

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Inward rectifier potassium (Kir) channels play important roles in the maintenance and control of cell excitability. Both intracellular trafficking and modulation of Kir channel activity are regulated by protein-protein interactions. We adopted a proteomics approach to identify proteins associated with Kir2 channels via the channel C-terminal PDZ binding motif. Detergent-solubilized rat brain and heart extracts were subjected to affinity chromatography using a Kir2.2 C-terminal matrix to purify channel-interacting proteins. Proteins were identified with multidimensional high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry, N-terminal microsequencing, and immunoblotting with specific antibodies. We identified eight members of the MAGUK family of proteins (SAP97, PSD-95, Chapsyn-110, SAP102, CASK, Dlg2, Dlg3, and Pals2), two isoforms of Veli (Veli-1 and Veli-3), Mint1, and actin-binding LIM protein (abLIM) as Kir2.2-associated brain proteins. From heart extract purifications, SAP97, CASK, Veli-3, and Mint1 also were found to associate with Kir2 channels. Furthermore, we demonstrate for the first time that components of the dystrophin-associated protein complex, including ␣1-, ␤1-, and ␤2-syntrophin, dystrophin, and dystrobrevin, interact with Kir2 channels, as demonstrated by immunoaffinity purification and affinity chromatography from skeletal and cardiac muscle and brain. Affinity pull-down experiments revealed that Kir2.1, Kir2.2, Kir2.3, and Kir4.1 all bind to scaffolding proteins but with different affinities for the dystrophin-associated protein complex and SAP97, CASK, and Veli. Immunofluorescent localization studies demonstrated that Kir2.2 co-localizes with syntrophin, dystrophin, and dystrobrevin at skeletal muscle neuromuscular junctions. These results suggest that Kir2 channels associate with protein complexes that may be important to target and traffic channels to specific subcellular locations, as well as anchor and stabilize channels in the plasma membrane.

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Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT

Cited by 284 publications

References 19 publications

Relative Quantification of Stable Isotope Labeled Peptides Using a Linear Ion Trap-Orbitrap Hybrid Mass Spectrometer

Relative Quantification of Stable Isotope Labeled Peptides Using a Linear Ion Trap-Orbitrap Hybrid Mass Spectrometer

Unraveling the Ubiquitome of the Human Malaria Parasite

Protein Trafficking and Anchoring Complexes Revealed by Proteomic Analysis of Inward Rectifier Potassium Channel (Kir2.x)-associated Proteins

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