Abstract：
:Dysregulated protein phosphorylation is a primary culprit in multiple physiopathological states. Hence, although analysis of signaling cascades on a proteome-wide scale would provide significant insight into both normal and aberrant cellular function, such studies are simultaneously limited by sheer biological complexity and concentration dynamic range. In principle, immobilized metal affinity chromatography (IMAC) represents an ideal enrichment method for phosphoproteomics. However, anecdotal evidence suggests that this technique is not widely and successfully applied beyond analysis of simple standards, gel bands, and targeted protein immunoprecipitations. Here, we report significant improvements in IMAC-based methodology for enrichment of phosphopeptides from complex biological mixtures. Moreover, we provide detailed explanation for key variables that in our hands most influenced the outcome of these experiments. Our results indicate 5- to 10-fold improvement in recovery of singly- and multiply phosphorylated peptide standards in addition to significant improvement in the number of high-confidence phosphopeptide sequence assignments from global analysis of cellular lysate. In addition, we quantitatively track phosphopeptide recovery as a function of phosphorylation state, and provide guidance for impedance-matching IMAC column capacity with anticipated phosphopeptide content of complex mixtures. Finally, we demonstrate that our improved methodology provides for identification of phosphopeptide distributions that closely mimic physiological conditions.
journal_name
J Proteome Resjournal_title
Journal of proteome researchauthors
Ndassa YM,Orsi C,Marto JA,Chen S,Ross MMdoi
10.1021/pr0602803subject
Has Abstractpub_date
2006-10-01 00:00:00pages
2789-99issue
10eissn
1535-3893issn
1535-3907journal_volume
5pub_type
杂志文章abstract：:Glycosylation is one of the most common post-translational modifications in proteins, existing in ~50% of mammalian proteins. Several research groups have demonstrated that mass spectrometry is an efficient technique for glycopeptide identification; however, this problem is still challenging because of the enormous di...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.5b00299
更新日期：2015-12-04 00:00:00
abstract：:Glycoproteins influence numerous indispensable biological functions, and changes in protein glycosylation have been observed in various diseases. The identification and characterization of glycoprotein and glycosylation sites by mass spectrometry (MS) remain challenging tasks, and great efforts have been devoted to th...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.6b00772
更新日期：2016-11-04 00:00:00
abstract：:Our cyanylation (CN)-based methodology for determining disulfide structure of cystinyl proteins overcomes the limitations of conventional proteolytic methods. However, the CN-based method has the potential drawback that occasionally some CN-induced cleavage fragments may not be detected. We show that CN-based methods ...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr049961t
更新日期：2004-07-01 00:00:00
abstract：:Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.5b00804
更新日期：2015-11-06 00:00:00
abstract：:A common animal model of chemical hepatocarcinogenesis was used to demonstrate the potential identification of carcinogenicity related protein signatures/biomarkers. Therefore, an animal study in which rats were treated with the known liver carcinogen N-nitrosomorpholine (NNM) or the corresponding vehicle was evaluate...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr050279o
更新日期：2006-02-01 00:00:00
abstract：:The HIV-1-associated neurocognitive disorder occurs in approximately one-third of infected individuals. It has persisted in the current era of antiretroviral therapy, and its study is complicated by the lack of biomarkers for this condition. Since the cerebrospinal fluid is the most proximal biofluid to the site of pa...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr800854t
更新日期：2009-05-01 00:00:00
abstract：:Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass ...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr200759m
更新日期：2012-02-03 00:00:00
abstract：:Quorum sensing (QS) in Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak, is mediated by the diffusible signal factor (DSF). DSF-mediating QS has been shown to control virulence and a set of virulence-related functions; however, the expression profiles and functions of extracellular pro...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr4001543
更新日期：2013-07-05 00:00:00
abstract：:Preeclampsia is a pregnancy-specific hypertensive syndrome and a major cause of maternal and fetal morbidity and mortality. At the present time, no reliable screening tests to identify women at risk are available. We have compared the amniotic fluids (AF) proteomic maps of five preeclamptic patients with those of five...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr060315z
更新日期：2007-01-01 00:00:00
abstract：:Sterols are membrane-bound isoprenoid lipids that are required for cell viability and growth. In plants, it is generally assumed that 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) is a key element of their biosynthesis, but the molecular regulation of that pathway is largely unknown. In an attempt to identify regula...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr201127u
更新日期：2012-02-03 00:00:00
abstract：:Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the ...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr400877e
更新日期：2013-12-06 00:00:00
abstract：:Although tandem mass spectrometry (MS/MS) has become an integral part of proteomics, intensity patterns in MS/MS spectra are rarely weighted heavily in most widely used algorithms because they are not yet fully understood. Here a knowledge mining approach is demonstrated to discover fragmentation intensity patterns an...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr070106u
更新日期：2008-01-01 00:00:00
abstract：:Low energy modes have been calculated for the largest possible number of available representatives (>150) of EF-hand domains belonging to different members of the calcium-binding EF-hand protein superfamily. These proteins are the major actors in signal transduction. The latter, in turn, relies on the dynamical proper...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr070314m
更新日期：2007-11-01 00:00:00
abstract：:The application of a data-independent acquisition (DIA) method ("SONAR") that employs a rapidly scanning quadrupole is described for the lipidomic analysis of complex biological extracts. Using this approach, the MS acquisition window can be varied between 1 and 25 Da, enabling the isolation of ions prior to their ent...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.9b00334
更新日期：2019-11-01 00:00:00
abstract：:We report the proteomic characterization and biological activities of the venom of the black-speckled palm pitviper, Bothriechis nigroviridis, a neotropical arboreal pitviper from Costa Rica. In marked contrast to other Bothriechis species investigated, the venom of B. nigroviridis does not possess detectable Zn(2+)-d...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr100545d
更新日期：2010-08-06 00:00:00
abstract：:Protein cleavage coupled with isotope dilution mass spectrometry (PC-IDMS) has the potential to provide the absolute concentration of a specific protein, or multiple proteins, in complex mixtures. However, PC-IDMS differs from standard IDMS since the internal standard is a different molecule than the analyte at the st...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr034124x
更新日期：2004-05-01 00:00:00
abstract：:Target-decoy approach (TDA) is the dominant strategy for false discovery rate (FDR) estimation in mass-spectrometry-based proteomics. One of its main applications is direct FDR estimation based on counting of decoy matches above a certain score threshold. The corresponding equations are widely employed for filtering o...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.6b00144
更新日期：2017-02-03 00:00:00
abstract：:A well-described animal model was used to understand the molecular mechanisms of carcinogenesis and metastasis of gastric cancer at the protein level. Gastric cancer was induced in 12 Wistar rats by oral administration of N-methyl-N'-Nitro-N-Nitrosoguanidine (MNNG). The protein expression patterns of normal gastric ti...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr049916l
更新日期：2004-09-01 00:00:00
abstract：:Mass spectrometry is the main analytical technique currently used to address the challenges of glycomics as it offers unrivalled levels of sensitivity and the ability to handle complex mixtures of different glycan variations. Determination of glycan structures from analysis of MS data is a major bottleneck in high-thr...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr7008252
更新日期：2008-04-01 00:00:00
abstract：:Protein phosphorylation is the most abundant reversible covalent modification. Human protein kinases participate in almost all biological pathways, and approximately half of the kinases are associated with disease. PhoSigNet was designed to store and display human phosphorylation-mediated signal transduction networks,...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.5b00249
更新日期：2015-07-02 00:00:00
abstract：:Glioblastoma multiforme (GBM) is the most aggressive malignant primary brain tumor, with a dismal mean survival even with the current standard of care. Although in vitro cell systems can provide mechanistic insight into the regulatory networks governing GBM cell proliferation and migration, clinical samples provide a ...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr500418w
更新日期：2014-11-07 00:00:00
abstract：:Whole cell protein and outer membrane protein (OMP) extracts were compared for their ability to differentiate and delineate the correct database organism to an experimental sample and for the degree of dissimilarity to the nearest neighbor database organism strains. These extracts were isolated from pathogenic and non...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr100402y
更新日期：2010-07-02 00:00:00
abstract：:The increasing occurrence of multidrug resistant tuberculosis exerts a major burden on treatment of this infectious disease. Thioridazine, previously used as a neuroleptic, is active against extensively drug resistant tuberculosis when added to other second- and third-line antibiotics. By quantitatively studying the p...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.5b01037
更新日期：2016-06-03 00:00:00
abstract：:Crohn's disease is an inflammatory disorder of the bowel, believed to arise from the dysregulation of intestinal mucosal immunity. The interleukin-10-deficient (IL10-/-) mouse, which develops intestinal inflammation in the presence of gut microflora, serves as a mouse model of Crohn's disease. Nontargeted urinary meta...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr800999t
更新日期：2009-04-01 00:00:00
abstract：:The peptide toxins in the venoms of small invertebrates such as stinging ants have rarely been studied due to the limited amount of venom available per individual. We used a venomics strategy to identify the molecular diversity of the venom peptidome for the myrmicine ant Tetramorium bicarinatum. The methodology inclu...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.8b00452
更新日期：2018-10-05 00:00:00
abstract：:Type 1 diabetes, in human patients and NOD mice, results from an immune attack on insulin-producing beta-cells of the pancreas by autoreactive T lymphocytes. In NOD mice, genetically controlled perturbations in the signaling pathways downstream of the antigen-specific T cell receptor (TCR) may be instrumental in the a...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr100035b
更新日期：2010-06-04 00:00:00
abstract：:Ceramides are central molecules in sphingolipid metabolism. They are involved in the regulation of cancer-cell growth, differentiation, senescence and apoptosis. To better understand how these secondary messengers induce their biological effects, adenocarcinoma cells (HCT116) were treated with exogenous long-chain cer...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr9005316
更新日期：2009-10-01 00:00:00
abstract：:Pancreatic cystic neoplasms represent 10-15% of primary cystic masses of the pancreas. While pancreatic cysts are detected with an increasing frequency due to the use of advanced imaging modalities in clinical practice, the diagnosis of pancreatic cystic neoplasms remains unsatisfactory because available diagnostic te...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr2000557
更新日期：2011-05-06 00:00:00
abstract：:Protein-protein control recognition remains a huge challenge, and its development depends on understanding the chemical and biological mechanisms by which these interactions occur. Here we describe a protein-protein control recognition technique based on the dominant electrostatic interactions occurring between the pr...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr100027k
更新日期：2010-06-04 00:00:00
abstract：:Herein, we report on the venom proteome of Vipera anatolica senliki, a recently discovered and hitherto unexplored subspecies of the critically endangered Anatolian meadow viper endemic to the Antalya Province of Turkey. Integrative venomics, including venom gland transcriptomics as well as complementary bottom-up and...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.9b00869
更新日期：2020-04-03 00:00:00
