MS1, MS2, and SQT—three unified, compact, and easily parsed file formats for the storage of shotgun proteomic spectra and identifications

McDonald, W. Hayes; Tabb, David L.; Sadygov, Rovshan G.; MacCoss, Michael J.; Venable, John D.; Graumann, Johannes; Johnson, J. R.; Cociorva, Daniel; Yates, John R.

doi:10.1002/rcm.1603

Cited by 346 publications

(316 citation statements)

References 15 publications

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“…Protein identification and quantification and analysis were done with Integrated Proteomics Pipeline -IP2 (Integrated Proteomics Applications, Inc., www.integratedproteomics.com/) using ProLuCID, DTASelect2, Census, and QuantCompare. Spectrum raw files were extracted into ms1 and ms2 files from raw files using RawExtract 1.9.9 (http://fields.scripps.edu/downloads.php) (103), and the tandem mass spectra were searched against the European Bioinformatic Institute (IPI) mouse protein database (www.ebi.ac.uk/IPI/IPImouse.html, downloaded on January 1, 2009). To estimate peptide probabilities and FDRs accurately, we used a target/decoy database containing the reversed sequences of all the proteins appended to the target database (104).…”

Section: Methodsmentioning

confidence: 99%

In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

Butko¹,

Savas

Friedman³

et al. 2013

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

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Postnatal bilateral whisker trimming was used as a model system to test how synaptic proteomes are altered in barrel cortex by sensory deprivation during synaptogenesis. Using quantitative mass spectrometry, we quantified more than 7,000 synaptic proteins and identified 89 significantly reduced and 161 significantly elevated proteins in sensory-deprived synapses, 22 of which were validated by immunoblotting. More than 95% of quantified proteins, including abundant synaptic proteins such as PSD-95 and gephyrin, exhibited no significant difference under high-and low-activity rearing conditions, suggesting no tissue-wide changes in excitatory or inhibitory synaptic density. In contrast, several proteins that promote mature spine morphology and synaptic strength, such as excitatory glutamate receptors and known accessory factors, were reduced significantly in deprived synapses. Immunohistochemistry revealed that the reduction in SynGAP1, a postsynaptic scaffolding protein, was restricted largely to layer I of barrel cortex in sensorydeprived rats. In addition, protein-degradation machinery such as proteasome subunits, E2 ligases, and E3 ligases, accumulated significantly in deprived synapses, suggesting targeted synaptic protein degradation under sensory deprivation. Importantly, this screen identified synaptic proteins whose levels were affected by sensory deprivation but whose synaptic roles have not yet been characterized in mammalian neurons. These data demonstrate the feasibility of defining synaptic proteomes under different sensory rearing conditions and could be applied to elucidate further molecular mechanisms of sensory development. mass spectrometric proteomics | somatosensory cortex | experience-dependent plasticity | synaptic protein dynamics S ensory information is encoded in the brain by activation of specific neuronal circuits that operate via chemical synapses. Important sensory experiences are stored by strengthening activated synapses in the circuit, a process known as "experience-dependent plasticity" (1). When animals are deprived of sensory experience during development, for example, by trimming rodent whiskers from birth to 30 d after birth, synaptic strength and mature synaptic morphology are attenuated, suggesting that the low activity in the deprived circuits interferes with normal development (2-7). However, the molecular changes that alter these synaptic properties in response to experience remain unclear. Synaptic activation has been shown to promote regulated and highly localized effects on synaptic proteins such as local translation, recruitment, and targeted degradation (8-11), and this spatiotemporal control of protein availability is required for long-term plasticity and synaptic maturation, which are fundamental in memory formation and storage and ultimately for an organism's survival (12-14). The need for highly controlled protein availability is highlighted in studies of neurological diseases, which often result from the disruption of protein availability at the synapse (15-17). ...

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Section: Methodsmentioning

confidence: 99%

In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

Butko¹,

Savas

Friedman³

et al. 2013

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

Self Cite

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“…Trichloroacetic acid-precipitated proteins were urea-denatured, reduced, alkylated, and digested with endoproteinase Lys-C (Roche Applied Science) followed by modified trypsin (Roche Applied Science) as described (40). Peptide mixtures were loaded onto 100-m fused silica microcapillary columns packed with 5-m C 18 reverse-phase (Aqua, Phenomenex), strong cation-exchange (PartiSphere SCX, Whatman), and reverse-phase particles (42). Loaded microcapillary columns were placed in-line with an Agilent 1100 series quaternary high pressure liquid chromatography pump and an LTQ ion trap mass spectrometer equipped with a nano-liquid chromatography-electrospray ionization source (Thermo Finnigan).…”

Section: Methodsmentioning

confidence: 99%

Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

Takahashi

Martin-Brown

Washburn

et al. 2009

Journal of Biological Chemistry

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Proteomic analyses have contributed substantially to our understanding of diverse cellular processes. Improvements in the sensitivity of mass spectrometry approaches are enabling more in-depth analyses of protein-protein networks and, in some cases, are providing surprising new insights into well established, longstanding problems. Here, we describe such a proteomic analysis that exploits MudPIT mass spectrometry and has led to the discovery of a physical and functional link between the orphan nuclear receptor hepatocyte nuclear factor 4␣ (HNF4␣) and transcription factor IID (TFIID). A systematic characterization of the HNF4␣-TFIID link revealed that the HNF4␣ DNA-binding domain binds directly to the TATA boxbinding protein (TBP) and, through this interaction, can target TBP or TFIID to promoters containing HNF4␣-binding sites in vitro. Supporting the functional significance of this interaction, an HNF4␣ mutation that blocks binding of TBP to HNF4␣ interferes with HNF4␣ transactivation activity in cells. These findings identify an unexpected role for the HNF4␣ DNA-binding domain in mediating key regulatory interactions and provide new insights into the roles of HNF4␣ and TFIID in RNA polymerase II transcription.Promoter-specific transcription by RNA polymerase II depends on the general initiation factors TFIIA, 2 -B, -D, -E, -F, and -H, which are the minimal set of factors needed for assembly of the preinitiation complex and subsequent initiation of transcription (1-4). The activity of RNA polymerase II and the general transcription factors is controlled by a host of DNAbinding transcription factors, the Mediator of RNA polymerase II transcription, and other coactivators and corepressors (5-7).Preinitiation complex assembly begins with DNA sequencespecific binding of TFIID to the core promoter to provide a nucleoprotein recognition site for subsequent binding of polymerase II and the remaining general transcription factors. The TATA box-binding protein (TBP) subunit of TFIID recognizes the TATA box element and can substitute for TFIID in transcription in vitro. Additional TBP-associated factor (TAF) subunits of TFIID recognize the initiator (Inr) and other core promoter elements and contribute to functional interactions between the general transcription machinery and DNA-binding transcription factors and coregulators (1,8,9). TBP has also been shown to be an essential component of SL1 and TFIIIB, which are multisubunit general transcription factors for RNA polymerases I and III, respectively (10 -16).Because of its central role in transcriptional regulation, there has been considerable interest in defining the repertoire of TBP-interacting proteins. Biochemical studies that led to definition of TFIID, SL1, TFIIIB, and other TBP-associated proteins have typically used conventional chromatography and/or immunoaffinity purification methods to purify TBP-containing complexes (17), followed by protein isolation by reverse-phase chromatography or SDS-PAGE and identification by Edman sequencing or mass spectrometry (e....

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“…Precursor ions were required to fall within 1.25 m/z of the position expected from their average masses, and fragment ions were required to fall within 0.5 m/z of their monoisotopic positions. The database searches produced raw identifications in SQT file format (spectral data SEQUEST search results) (41).…”

Section: Lc-ms-ms Analyses-mentioning

confidence: 99%

Global Analysis of Protein Damage by the Lipid Electrophile 4-Hydroxy-2-nonenal

Codreanu

Zhang

Sobecki

et al. 2009

Molecular & Cellular Proteomics

138

163

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Lipid peroxidation yields a variety of electrophiles, which are thought to contribute to the molecular pathogenesis of diseases involving oxidative stress, yet little is known of the scope of protein damage caused by lipid electrophiles. We identified protein targets of the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE) in RKO cells treated with 50 or 100 M HNE. HNE Michael adducts were biotinylated by reaction with biotinamidohexanoic acid hydrazide, captured with streptavidin, and the captured proteins were resolved by one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and identified by liquid chromatography-tandem mass spectrometry. Of the 1500؉ proteins identified, 417 displayed a statistically significant increase in adduction with increasing HNE exposure concentration. We further identified 18 biotin hydrazide-modified, HNE-adducted peptides by specific capture using anti-biotin antibody and analysis by high resolution liquid chromatography-tandem mass spectrometry. A subset of the identified HNE targets were validated with a streptavidin capture and immunoblotting approach, which enabled detection of adducts at HNE exposures as low as 1 M. Protein interaction network analysis indicated

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MS1, MS2, and SQT—three unified, compact, and easily parsed file formats for the storage of shotgun proteomic spectra and identifications

Cited by 346 publications

References 15 publications

In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

Global Analysis of Protein Damage by the Lipid Electrophile 4-Hydroxy-2-nonenal

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