A Data Processing Pipeline for Mammalian Proteome Dynamics Studies Using Stable Isotope Metabolic Labeling

Guan, Shenheng; Price, John C.; Prusiner, Stanley B.; Ghaemmaghami, Sina; Burlingame, Alma L.

doi:10.1074/mcp.m111.010728

Cited by 137 publications

(162 citation statements)

References 23 publications

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“…Data Processing-Peaklists were generated with PAVA, an inhouse software (35). Database searches were performed using Protein Prospector (version 5.9.0).…”

Section: Methodsmentioning

confidence: 99%

N- and O-Glycosylation in the Murine Synaptosome

Trinidad

Schoepfer²,

Burlingame

et al. 2013

Molecular & Cellular Proteomics

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We present the first large scale study characterizing both N-and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N-and O-linked carbohydrate structures. Liquid chromatography-MS (LC/ MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N-and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues. Molecular & Cellular

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“…Data Processing-Peaklists were generated with PAVA, an inhouse software (35). Database searches were performed using Protein Prospector (version 5.9.0).…”

Section: Methodsmentioning

confidence: 99%

N- and O-Glycosylation in the Murine Synaptosome

Trinidad

Schoepfer²,

Burlingame

et al. 2013

Molecular & Cellular Proteomics

Self Cite

163

230

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“…L and H formed two separate populations by a nonnegative least square approach as described previously (Guan et al, 2011;Nelson et al, 2014). All filters used for partial 15 N labeling were kept (Nelson et al, 2014) and two more filters were applied: First, a minimum 80% 15 N enrichment was required to disregard quantifications with high noise level between L and H; second, a minimum signal-to-noise ratio of 5 was used to disregard high neighboring noise contribution to L and H. A total of 432 Agilent .d files were further processed by Trans Proteomic Pipeline to get protein probabilities for identification.…”

Section: Determining Changes In Specific Protein Abundance Over 5 D Umentioning

confidence: 99%

Protein Degradation Rate in Arabidopsis thaliana Leaf Growth and Development

et al. 2017

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We applied 15 N labeling approaches to leaves of the Arabidopsis thaliana rosette to characterize their protein degradation rate and understand its determinants. The progressive labeling of new peptides with 15 N and measuring the decrease in the abundance of >60,000 existing peptides over time allowed us to define the degradation rate of 1228 proteins in vivo. We show that Arabidopsis protein half-lives vary from several hours to several months based on the exponential constant of the decay rate for each protein. This rate was calculated from the relative isotope abundance of each peptide and the fold change in protein abundance during growth. Protein complex membership and specific protein domains were found to be strong predictors of degradation rate, while N-end amino acid, hydrophobicity, or aggregation propensity of proteins were not. We discovered rapidly degrading subunits in a variety of protein complexes in plastids and identified the set of plant proteins whose degradation rate changed in different leaves of the rosette and correlated with leaf growth rate. From this information, we have calculated the protein turnover energy costs in different leaves and their key determinants within the proteome.

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“…The parameters for peptide identification were trypsin cleavage specificity with 1 missed cleavage allowed, 0 non-tryptic termini, monoisotopic precursors, 20-ppm precursor maximum mass error, 0.6-Da fragment ion maximum mass error, ϩ2 or ϩ3 precursor charge state, fixed carbamidomethylation of Cys residues, variable deamidation of Asn and Gln residues, variable oxidation of Met residues, and variable conversion of peptide N-terminal Gln residues to pyroglutamic acid. Following Batch-Tag, a list of identified GFAP peptides was generated using the Search Compare module, and subsequent data analysis was performed using a data processing pipeline for mammalian proteome dynamics studies created by S. Guan and coworkers (25,28).…”

Section: Mass Spectrometry and Data Analysis Of Silam Samplesmentioning

confidence: 99%

“…Computational data analysis was performed using a series of previously described data processing modules (25,28 Figure 1. Peptides used for in vitro and in vivo GFAP kinetic studies.…”

Section: Gfapmentioning

confidence: 99%

Glial fibrillary acidic protein exhibits altered turnover kinetics in a mouse model of Alexander disease

Moody

Barrett-Wilt

Sussman

et al. 2017

Journal of Biological Chemistry

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Edited by George N. DeMartinoMutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP) lead to the rare and fatal disorder, Alexander disease (AxD). A prominent feature of the disease is aberrant accumulation of GFAP. It has been proposed that this accumulation occurs because of an increase in gene transcription coupled with impaired proteasomal degradation, yet this hypothesis remains untested. We therefore sought to directly investigate GFAP turnover in a mouse model of AxD that is heterozygous for a disease-causing point mutation (Gfap R236H/؉ ) (and thus expresses both wild-type and mutant protein). Stable isotope labeling by amino acids in cell culture, using primary cortical astrocytes, indicated that the in vitro halflives of total GFAP in astrocytes from wild-type and mutant mice were similar at ϳ3-4 days. Surprisingly, results obtained with stable isotope labeling of mammals revealed that, in vivo, the half-life of GFAP in mutant mice (15.4 ؎ 0.5 days) was much shorter than that in wild-type mice (27.5 ؎ 1.6 days). These unexpected in vivo data are most consistent with a model in which synthesis and degradation are both increased. Our work reveals that an AxD-causing mutation alters GFAP turnover kinetics in vivo and provides an essential foundation for future studies aimed at preventing or reducing the accumulation of GFAP. In particular, these data suggest that elimination of GFAP might be possible and occurs more quickly than previously surmised.Alexander disease (AxD) 2 is a rare and often fatal human disease of the central nervous system caused by dominant mutations in the astrocyte intermediate filament glial fibrillary acidic protein (GFAP) (1). Most AxD patients have de novo mutations in GFAP (2), encoding for various missense mutations as well as small in-frame insertions and deletions. The hallmark feature of the pathology is the formation of aggregates, known as Rosenthal fibers (RFs), within the cell bodies and processes of astrocytes, along with variable degrees of white matter deficits. Although the genetic basis for AxD is clear, the mechanisms by which GFAP mutations lead to astrocyte dysfunction and the cascade of secondary effects on other cells in the central nervous system remain unresolved. Previous studies demonstrated that simple overexpression of wild-type GFAP to high levels induces the formation of RFs (3), and indeed increased levels of GFAP are consistently present in autopsy samples from patients with AxD (4 -8). Mouse models engineered to express mutations equivalent to common human mutations (9) illustrate a connection between GFAP levels and severity of disease, which has led to the concept of "GFAP toxicity."The excessive accumulation of GFAP and the formation of RFs in AxD presumably reflect a fundamental alteration in proteostasis. Proteostasis, or protein homeostasis, involves a vast network of pathways that control protein synthesis, folding, trafficking, aggregation, and degradation. Protein aggregation disorders such as Alzhei...

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A Data Processing Pipeline for Mammalian Proteome Dynamics Studies Using Stable Isotope Metabolic Labeling

Cited by 137 publications

References 23 publications

N- and O-Glycosylation in the Murine Synaptosome

N- and O-Glycosylation in the Murine Synaptosome

Protein Degradation Rate in Arabidopsis thaliana Leaf Growth and Development

Glial fibrillary acidic protein exhibits altered turnover kinetics in a mouse model of Alexander disease

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