Dataset Information

Improved plasma membrane proteome coverage with a label-free non-affinity-purified workflow

ABSTRACT: The proteins of the cellular plasma membrane perform important functions relating to homeostasis and intercellular communication. Due to its overall low cellular abundance, amphipathic character, and low membrane-to-cytoplasm ratio, the plasma membrane proteome has been challenging to isolate and characterize, and is poorly represented in standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. In this study, we employ a label-free, non-affinity-purified workflow for the enrichment of the plasma membrane proteome and use subsequent bioinformatics tools to select plasma membrane proteins, herein referred to as the surfaceome. Using this methodology, we identified over 2500 cell surface-associated proteins in a human acute myeloid leukemia (AML) cell line. These surface proteins comprised 60% of all detected cellular proteins, a number that substantially exceeds the depth of coverage in previously published studies describing the leukemia surfaceome.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Leukocyte, Cell Culture

DISEASE(S): Acute Leukemia

SUBMITTER: Steven Seeholzer

LAB HEAD: Steven H. Seeholzer

PROVIDER: PXD004801 | Pride | 2017-01-31

REPOSITORIES: Pride

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Improved surfaceome coverage with a label-free nonaffinity-purified workflow.

Glisovic-Aplenc Tina T Gill Saar S Spruce Lynn A LA Smith Ian R IR Fazelinia Hossein H Shestova Olga O Ding Hua H Tasian Sarah K SK Aplenc Richard R Seeholzer Steven H SH

Proteomics 20170306 7

The proteins of the cellular plasma membrane (PM) perform important functions relating to homeostasis and intercellular communication. Due to its overall low cellular abundance, amphipathic character, and low membrane-to-cytoplasm ratio, the PM proteome has been challenging to isolate and characterize, and is poorly represented in standard LC-MS/MS analyses. In this study, we employ sucrose gradient ultracentrifugation for the enrichment of the PM proteome, without chemical labeling and affinity ...[more]

PMID: 28116781

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Project description:Previous systems-based proteomic approaches have characterized alterations in protein co-expression networks of unfractionated asymptomatic (AsymAD) and symptomatic Alzheimer’s disease (AD) brains. However, it remains unclear how sample fractionation and sub-proteomic analysis influences the organization of these protein networks and their relationship to clinicopathological traits of disease. In this proof-of-concept study, we performed a systems-based sub-proteomic analysis of membrane-enriched post-mortem brain samples from pathology-free control, AsymAD, and AD brains (n=6 per group). Label-free mass spectrometry based on peptide ion intensity was used to quantify the 18 membrane-enriched fractions. Differential expression and weighted protein co-expression network analysis (WPCNA) were then used to identify and characterize modules of co-expressed proteins most significantly altered between the groups. We identified a total of 27 modules of co-expressed membrane-associated proteins. In contrast to the unfractionated proteome, these networks did not map strongly to cell-type specific markers. Instead, these modules were principally organized by their associations with a wide variety of membrane-bound compartments and organelles. Of these, the mitochondrion was associated with the greatest number of modules, followed by modules linked to the cell surface compartment. In addition, we resolved networks with strong associations to the endoplasmic reticulum, Golgi apparatus, nucleus, and other membrane-bound organelles. Fourteen of the 27 modules demonstrated significant correlations with clinical and pathological AD phenotypes. These results revealed that the proteins within individual compartments feature a heterogeneous array of AD-associated expression patterns, particularly during preclinical stages of disease. In conclusion, this systems-based analysis of the membrane-associated AsymAD brain proteome yielded a unique network organization highly linked to cellular compartmentalization. Further study of this membrane-associated proteome may reveal novel insight into the complex pathways governing the earliest stages of disease.

Dataset Information

Improved plasma membrane proteome coverage with a label-free non-affinity-purified workflow

Publications

Improved surfaceome coverage with a label-free nonaffinity-purified workflow.

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