Off-Line High-pH Reversed-Phase Fractionation for In-Depth Phosphoproteomics

Batth, Tanveer S.; Francavilla, Chiara; Olsen, Jesper V.

doi:10.1021/pr500893m

Cited by 273 publications

(254 citation statements)

References 38 publications

Supporting

Mentioning

253

Contrasting

Order By: Relevance

“…Proteolytic digestion products were desalted on a Sep-Pak C 18 cartridge (Waters, Milford), lyophilized for 2 days and enriched in phosphopeptides by consecutive incubations with TiO 2 beads (38 -39). Half sample of each fraction was directly analyzed by LC-MS/MS whereas the remaining halves were pooled together and further fractionated using high pH fractionation prior MS analysis (40).…”

Section: Reagentsmentioning

confidence: 99%

Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes

Osinalde

Mitxelena

Sanchez-Quiles

et al. 2016

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2-dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4 ؉ T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer The underlying principle of cancer immunotherapy is to eliminate malignant cells by tuning the immune system (1-2). This revolutionary way of fighting tumors was originated three decades ago when a patient suffering from metastatic melanoma was treated with the T-cell growth promoting factor interleukin-2 (IL-2) 1 (3). The success of IL-2 administration in fighting metastatic melanoma demonstrated for the first time that solely potentiating the activation of T lymphocytes could abrogate certain human cancers (4). Current immunotherapy approaches include the use of autologous gene-engineered T cells that, once expanded ex vivo with IL-2, are re-infused back into patients by the so-called adoptive cell transfer therapy (ACT) (5-7). Despite the promising results of this approach, a safe and long-lasting expansion of transferred T cells remains a major challenge because of the undesirable side effects derived from the use of IL-2. Continued exposure to high doses of IL-2 results in increased susceptibility of T cells to apoptosis (8). Moreover, IL-2 is also a critical component for regulatory T-cell (T reg ) development and function (9 -10), and as such it functions as a negative regulator of the immune response (11). Consequently, although IL-2 constitutes a key component of current immunotherapies, a great deal of effort is being devoted to the development of novel strategies that would boost the T-cell immune response more safely. In this regard, it has been shown that IL-2-related toxicity can be partially minimized by the use of gene-engineered T cells ex...

show abstract

Section: Reagentsmentioning

confidence: 99%

Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes

Osinalde

Mitxelena

Sanchez-Quiles

et al. 2016

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…However, recent protocols either avoid this step (Soufi et al 2015) or use alternative peptide fractionation methods, such as high-pH reversed-phase chromatography (Batth et al 2014). If you are applying an alternative prefractionation protocol, proceed to Step 15 for TiO 2 enrichment of peptide fractions.…”

Section: Performing Scx Chromatographymentioning

confidence: 99%

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-Based Quantitative Proteomics and Phosphoproteomics in Fission Yeast

Carpy

Koch

Bicho

et al. 2017

Cold Spring Harb Protoc

View full text Add to dashboard Cite

Modern mass spectrometry (MS)-based approaches are capable of identifying and quantifying thousands of proteins and phosphorylation events in a single biological experiment. Here we present a (phospho)proteomic workflow based on in-solution proteome digestion of samples labeled by stable isotope labeling by amino acids in cell culture (SILAC) and phosphopeptide enrichment using strong cation exchange (SCX) and TiO 2 chromatographies. These procedures are followed by high-accuracy MS measurement on an Orbitrap mass spectrometer and subsequent bioinformatic processing using MaxQuant software. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol.RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. ReagentsAcetonitrile (

show abstract

“…In addition, because of their low abundance and ionization properties (34), few phosphopeptides can be quantified by direct MRM analysis of neat cellular lysate, and enrichment is required. Although this can be achieved using the multidimensional biochemical fractionation workflows (10,33,35), these workflows require specialized expertise/instrumentation, are low in throughput, and are cost-prohibitive as a workhorse assay platform for the community. We have greatly simplified sample preparation, reduced assay costs, and improved throughput by coupling immunoaffinity enrichment of peptides with quantification by MRM (28).…”

Section: Selection Of Analytes and Development Of Assaymentioning

confidence: 99%

Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling

Whiteaker

Zhao

Yan

et al. 2015

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

In most cell signaling experiments, analytes are measured one Western blot lane at a time in a semiquantitative and often poorly specific manner, limiting our understanding of network biology and hindering the translation of novel therapeutics and diagnostics. We show the feasibility of using multiplex immuno-MRM for phospho-pharmacodynamic measurements, establishing the potential for rapid and precise quantification of cell signaling networks. A 69-plex immuno-MRM assay targeting the DNA damage response network was developed and characterized by response curves and determinations of intra-and interassay repeatability. The linear range was >3 orders of magnitude, the median limit of quantification was 2.0 fmol/mg, the median intra-assay variability was 10% CV, and the median interassay variability was 16% CV. The assay was applied in proof-of-concept studies to immortalized and primary human cells and surgically excised cancer tissues to quantify exposure-response relationships and the effects of a genomic variant (ATM kinase mutation) or pharmacologic ( Because there is limited correlation between mRNA and protein levels/activity (1), quantification of proteins and posttranslational modifications is critical to understanding cellular signaling and determining pharmacodynamic (PD) 1 responses. Phosphorylation is a key post-translational modification used in signaling networks to modulate protein/pathway activity, protein interactions, and protein localization in response to extracellular and intracellular stimuli. Many diseases exhibit dysfunctions in signaling networks, and thus major efforts to identify novel drug targets (e.g. kinase inhibitors) are based on signal transduction pathways (2).Currently the research community lacks high throughput, quantitative tools for studying phospho-signaling networks, hindering our basic understanding of network biology and hence the translation of novel therapeutics and companion diagnostics. In most experiments, one analyte is measured one Western blot lane at a time in a semiquantitative and often nonspecific manner. These drawbacks limit our ability to extend knowledge beyond individual phosphorylation events to a system-wide study of phosphorylation dynamics, which is critical because signal transduction pathways act as interconnected networks, and the effects of mutations in individual genes (as well as the effects of pharmacologic compounds) spread throughout the network (3). Although Western blotting and related traditional immuno-assay platforms (e.g. ELISA) have been pushed brilliantly to their limits and have formed the basis of many advances in biomedical research, they are inadequate to support the needs of the postgenomic world, in which we need innovative technologies for determining the effects of any experimental condition (e.g. agonist or antagonist exposures, genetic variations) on the major signal transduction networks of the human cell, using precise, standardized, moderate-to-high throughput methods that can be reproduced across laboratories.Newer tec...

show abstract

Off-Line High-pH Reversed-Phase Fractionation for In-Depth Phosphoproteomics

Cited by 273 publications

References 38 publications

Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes

Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-Based Quantitative Proteomics and Phosphoproteomics in Fission Yeast

Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling

Contact Info

Product

Resources

About