Quantitative Analysis of Brain Nuclear Phosphoproteins Identifies Developmentally Regulated Phosphorylation Events

Liao, Lujian; McClatchy, Daniel B.; Park, Sung Kyu; Xu, Tao; Lu, Bingwen; Yates, John R.

doi:10.1021/pr8003198

Cited by 42 publications

(41 citation statements)

References 75 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Two studies have analyzed phosphoproteomes of lower multicellular organisms, nematode larva (25) and barnacle (26); both used two-dimensional gel electrophoresis and found interesting stage-specific, as well as constitutive, protein phosphorylation. Similar analyses have been carried out in mammals, with protein phosphorylation dynamics being analyzed during early stages of embryonic stem cell development (27) and in brain nuclear proteins (28). Other studies in mammalian cells monitored time-dependent changes in protein phosphorylation abundance in response to epidermal growth factor stimulation (29,30), observing that for multiply phosphorylated proteins, different sites showed different dynamics trends.…”

Section: Discussionmentioning

confidence: 82%

Regulation Dynamics of Leishmania Differentiation: Deconvoluting Signals and Identifying Phosphorylation Trends

Tsigankov

Gherardini

Helmer-Citterich

et al. 2014

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Leishmania are obligatory intracellular parasitic protozoa that cause a wide range of diseases in humans, cycling between extracellular promastigotes in the mid-gut of sand flies and intracellular amastigotes in the phagolysosomes of mammalian macrophages. Although many of the molecular mechanisms of development inside macrophages remain a mystery, the development of a host-free system that simulates phagolysosome conditions (37°C and pH 5.5) has provided new insights into these processes. The time course of promastigote-to-amastigote differentiation can be divided into four morphologically distinct phases: I, signal perception (0 -5 h after exposure); II, movement cessation and aggregation (5-10 h); III, amastigote morphogenesis (10 -24 h); and IV, maturation (24 -120 h). Transcriptomic and proteomic analyses have indicated that differentiation is a coordinated process that results in adaptation to life inside phagolysosomes. Recent phosphoproteomic analysis revealed extensive differences in phosphorylation between promastigotes and amastigotes and identified stage-specific phosphorylation motifs. We hypothesized that the differentiation signal activates a phosphorylation pathway that initiates Leishmania transformation, and here we used isobaric tags for relative and absolute quantitation to interrogate the dynamics of changes in the phosphorylation profile during Leishmania donovani promastigote-to-amastigote differentiation. Analysis of 163 phosphopeptides (from 106 proteins) revealed six distinct kinetic profiles; with increases in phosphorylation predominated during phases I and III, whereas phases II and IV were characterized by greater dephosphorylation. Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. signalspecific; 37°C and pH 5.5), but not after either of the physical parameters separately. Several other protein kinases (including regulatory subunits) and phosphatases also showed changes in phosphorylation during differentiation. This work constitutes the first genome-scale interrogation of phosphorylation dynamics in a parasitic protozoa, revealing the outline of a signaling pathway during Leishmania differentiation.

show abstract

Section: Discussionmentioning

confidence: 82%

Regulation Dynamics of Leishmania Differentiation: Deconvoluting Signals and Identifying Phosphorylation Trends

Tsigankov

Gherardini

Helmer-Citterich

et al. 2014

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…For intertissue comparison, Trinidad et al (12) applied strong cation exchange (SCX) 1 and a titanium dioxide (TiO 2 ) column combined with iTRAQ to compare relative protein expression and phosphorylation status of murine cortex, midbrain, cerebellum, and hippocampus. Using 15 N-labeled rat brain as an internal standard, Yates and co-workers (13) have quantified phosphopeptides from p1 and p45 rat brain cortices and observed phosphorylation regulated distinctly on different sites compared with the protein level for a set of proteins. Whereas for the investigation of cells, Munton et al (14) used isobaric peptide tags to determine the absolute quantity of both phosphorylated and unphosphorylated peptides of candidate proteins.…”

mentioning

confidence: 99%

Concurrent Quantification of Proteome and Phosphoproteome to Reveal System-wide Association of Protein Phosphorylation and Gene Expression

Dai

Yang

et al. 2009

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Reversible phosphorylation of proteins is an important process modulating cellular activities from upstream, which mainly involves sequential phosphorylation of signaling molecules, to downstream where phosphorylation of transcription factors regulates gene expression. In this study, we combined quantitative labeling with multidimensional liquid chromatography-mass spectrometry to monitor the proteome and phosphoproteome changes in the initial period of adipocyte differentiation. The phosphorylation level of a specific protein may be regulated by a kinase or phosphatase without involvement of gene expression or as a phenomenon that accompanies the alteration of its gene expression. Concurrent quantification of phosphopeptides and nonphosphorylated peptides makes it possible to differentiate cellular phosphorylation changes at these two levels. Furthermore, on the system level, certain proteins were predicted as the targeted gene products regulated by identified transcription factors. Among them, several proteins showed significant expression changes along with the phosphorylation alteration of their transcription factors. This is to date the first work to concurrently quantify proteome and phosphoproteome changes during the initial period of adipocyte differentiation, providing an approach to reveal the system-wide association of protein phosphorylation and gene expression. Protein phosphorylation has been considered as a central role for cell regulation and signaling transduction. It is estimated that one-third of eukaryotic proteins are phosphorylated (1). In eukaryotes, the residues that undergo protein phosphorylation are mainly serine, threonine, and to a lesser extent tyrosine (2). Reversible phosphorylation of proteins is an important process for modulating cellular activities. At the upstream level, regulation of signaling transduction mainly relies on the appropriate phosphorylation events, such as sequential phosphorylation of various signaling molecules. At the downstream level, however, signaling transduction in the nucleus generally involves transcription of certain genes. Transcription factor activity, besides being regulated at the level of gene expression, is prominently regulated via post-translational events such as protein phosphorylation, processing, or localization (3). Phosphorylation or dephosphorylation of transcription-associated factors plays a crucial role in transcriptional regulation (see Fig. 1A).Most of the previous studies have focused on the initial period of signaling transduction, which mainly refers to the dynamic changes of phosphorylation events only. Recent developments in mass spectrometry promise to provide novel insights into the dynamics of protein expression and activities regulated by post-translational modifications (7,8). However, the isolation of phosphoproteins from complex mixtures and the determination of phosphorylation sites still remain a challenge. Different strategies have been applied for enrichment of phosphorylated peptides and proteins, including immuno...

show abstract

“…Yates and his coworkers demonstrated the strategy by using a 15 N-labeled rat in several differential proteomic and phosphoprotein analyses in various tissues like liver, brain, etc. [21][22][23][24] SILAC mouse or stable isotope-labeling in mammals has been used for quantitative analysis of proteomes from mice to determine protein functions under complex in vivo conditions. The F2 generation of mice fed with a diet containing heavy lysine was found to be completely labeled in all organs tested by Kruger and colleagues.…”

Section: Mirza Quantitative Ms-based Methods O3mentioning

confidence: 99%

Quantitative Mass Spectrometry-Based Approaches in Cardiovascular Research

Mirza¹

2012

Circ Cardiovasc Genet

View full text Add to dashboard Cite

ProteomicsC ardiovascular proteomics is a rapidly growing field, especially with the emergence of high-throughput mass spectrometry-based techniques. After the identification of proteins in a given sample, researchers are keen to find out how much of the protein is present, especially in a disease or a given state. Though challenging, over the past few years, several approaches have been established to quantify proteins from cells or tissues at a given state. In this review, we focus on mass spectrometry -based quantitative approaches for both large-scale and targeted proteomic analyses, which can be used in cardiovascular research. Several strategies using labeling and label-free approaches for both relative and absolute quantification (AQUA) have been established to meet the requirements of the researcher. Each of these methods has its own merits and limitations. We will discuss the methods that researchers can opt for to address the biological questions related to cardiovascular research.Cardiovascular proteomics is the branch of proteomics specifically applied to heart and vasculature. The field of proteomics has recently gained greater importance based on its potential to unravel the complex physiological and biochemical mechanisms and pathways in organisms and cells at the molecular level. The goal is to identify proteins that generate/mediate disease process, thereby, opening avenues to new diagnostics and therapeutic strategies. Proteins are much closer to the actual disease process, in most cases, than parent genes. They are the ultimate regulators of cell functions, and the vast majorities serve as drug targets. Differential protein expression analysis, by measuring the upregulated or downregulated proteins in disease state as compared with the normal healthy condition, is vital to understand the disease mechanism.In conjunction with various separation techniques, mass spectrometry (MS)-based methods evolved as the best techniques for the comprehensive characterization of proteins. 1,2After the initial protein identification, a more challenging approach in proteomics is the quantification of the proteins identified.3 Various methods of quantification by MS have been developed recently, for both relative and absolute measurements (Figure 1), and the technology is still expanding to find better and more efficient quantification approaches. This, in turn, is facilitated by the advancements in the MS instrumentation, including powerful analyzers and fragmentation technology. Most of the quantification methods are based on measurements at peptide level and fall under the bottom-up proteomics approach. In this review article, we will outline various MS-based approaches available for measuring the variation in protein abundances and how these techniques are playing a key role in cardiovascular research. Gel-Based QuantificationSeparation of proteins by polyacrylamide gel electrophoresis, followed by MS analysis, has been a common practice in proteomics. With the advent of 2D gel electrophoresis, quantificatio...

show abstract

Quantitative Analysis of Brain Nuclear Phosphoproteins Identifies Developmentally Regulated Phosphorylation Events

Cited by 42 publications

References 75 publications

Regulation Dynamics of Leishmania Differentiation: Deconvoluting Signals and Identifying Phosphorylation Trends

Regulation Dynamics of Leishmania Differentiation: Deconvoluting Signals and Identifying Phosphorylation Trends

Concurrent Quantification of Proteome and Phosphoproteome to Reveal System-wide Association of Protein Phosphorylation and Gene Expression

Quantitative Mass Spectrometry-Based Approaches in Cardiovascular Research

Contact Info

Product

Resources

About