Highly Efficient Depletion Strategy for the Two Most Abundant Erythrocyte Soluble Proteins Improves Proteome Coverage Dramatically

Ringrose, Jeffrey H.; Solinge, Wouter W. van; Mohammed, Shabaz; O’Flaherty, Martina; Wijk, Richard van; Heck, Albert J. R.; Slijper, Monique

doi:10.1021/pr8001029

Cited by 72 publications

(77 citation statements)

References 20 publications

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“…This disease model demonstrates progressive late-onset neurodegeneration accompanied by pathological changes and ␣-syn inclusion formation in selective brain regions such as the brainstem, whereas other regions such as the cortex remain largely unaffected (21). Frontal cortex and brainstem were dissected from A53T transgenic mice (30) and nontransgenic littermate control mice at 6 months of age (no apparent motor phenotype at the time that the mice were sacrificed).…”

Section: Methodsmentioning

confidence: 99%

α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

Fauvet

Mbefo

Fares

et al. 2012

Journal of Biological Chemistry

516

497

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Background:The oligomeric state of ␣-syn in vivo remains unknown. Results: ␣-syn in the CNS and produced by erythrocytes, mammalian cells, and Escherichia coli exists predominantly as a disordered monomer. Conclusion: Native ␣-syn from various sources behaves as unstructured and monomeric. Significance: Stabilizing monomeric ␣-syn, lowering its levels, and/or inhibiting its fibrillization remain viable therapeutic strategies for Parkinson disease.

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Section: Methodsmentioning

confidence: 99%

α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

Fauvet

Mbefo

Fares

et al. 2012

Journal of Biological Chemistry

516

497

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“…The top ten significantly increased and decreased peptides between high versus low hemolysis conditions are detailed in Table II. The majority of top peptides significantly increased in highly hemolyzed blood plasma correspond to different hemoglobin isoforms (Hba-a1, Q9CY10, Hbb-b1, A8DUK4, and Hba, P01942), a protein group comprising over 97% of the red blood cell proteome (37). The top ten peptides significantly decreased exhibited greater complexity and corresponded to proteins previously described as being localized to blood plasma, such as the copper-binding glycoprotein ceruloplasmin (Cp, Q61147) (38) a member of the immune-associated complement system, complement factor D (Cfd, P03953) (38 -39), as well as the lipoprotein scavenger apolipoprotein E (ApoE, P08226) (38 -39).…”

Section: Differential Analysis Of a Synaptic Density Fraction From Homentioning

confidence: 99%

Maximizing Peptide Identification Events in Proteomic Workflows Using Data-Dependent Acquisition (DDA)

Bateman

Goulding

Shulman

et al. 2014

Molecular & Cellular Proteomics

107

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Current analytical strategies for collecting proteomic data using data-dependent acquisition (DDA) are limited by the low analytical reproducibility of the method. Proteomic discovery efforts that exploit the benefits of DDA, such as providing peptide sequence information, but that enable improved analytical reproducibility, represent an ideal scenario for maximizing measureable peptide identifications in "shotgun"-type proteomic studies. Therefore, we propose an analytical workflow combining DDA with retention time aligned extracted ion chromatogram (XIC) areas obtained from high mass accuracy MS1 data acquired in parallel. We applied this workflow to the analyses of sample matrixes prepared from mouse blood plasma and brain tissues and observed increases in peptide detection of up to 30.5% due to the comparison of peptide MS1 XIC areas following retention time alignment of co-identified peptides. Furthermore, we show that the approach is quantitative using peptide standards diluted into a complex matrix. These data revealed that peptide MS1 XIC areas provide linear response of over three orders of magnitude down to low femtomole (fmol) levels. These findings argue that augmenting "shotgun" proteomic workflows with retention time alignment of peptide identifications and comparative analyses of corresponding peptide MS1 XIC areas improve the analytical performance of global proteomic discovery methods using DDA. Molecular & Cellular Proteomics 13: 10.1074/ mcp.M112.026500, 329-338, 2014.Label-free methods in mass spectrometry-based proteomics, such as those used in common "shotgun" proteomic studies, provide peptide sequence information as well as relative measurements of peptide abundance (1-3). A common data acquisition strategy is based on data-dependent acquisition (DDA) 1 where the most abundant precursor ions are selected for tandem mass spectrometry (MS/MS) analysis (1-2). DDA attempts to minimize redundant peptide precursor selection and maximize the depth of proteome coverage (2). However, the analytical reproducibility of peptide identifications obtained using DDA-based methods result in Ͻ75% overlap between technical replicates (3-4). Comparisons of peptide identifications between replicate analyses have shown that the rate of new peptide identifications increases sharply following two replicate sample injections and gradually tapers off after approximately five replicate injections (4). This phenomenon is due, in part, to the semirandom sampling of peptides in a DDA experiment (5).Alternate label-free methods focused on measuring the abundance of intact peptide ions, such as the accurate mass and time tag (AMT) approach (6 -8, 42), are aimed at differential analyses of extracted ion chromatogram (XIC) areas integrated from high mass accuracy peptide precursor mass spectra (MS1 spectra) exhibiting discrete chromatographic elution times. This method is particularly powerful for the analysis of post-translationally modified (PTM) peptides as pairing the low abundance of PTM candidates with the variabl...

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“…The interference of hemoglobin when detecting lowabundance proteins in the cytosol by LC-MS/MS analysis has been recently reported [15]. In the present work, we evaluate the influence of hemoglobin on 2-DE cytosolic patterns and we propose an alternative for hemoglobin depletion to those reported in the literature, i.e.…”

Section: Red Blood Cell Cytosolic Proteomementioning

confidence: 95%

“…5). They corresponded to peroxiredoxin-2, flavin reductase, carbonic anhydrase 1 and 2, G3PDH and catalase which are proteins of high abundance in erythrocytes cytosol [14,15]. No additional proteins could be detected in those bands; thus the possibility to unselectively remove minor proteins with this depletion methodology and miss them in 2-DE analysis is minimal.…”

Section: Red Blood Cell Cytosolic Proteomementioning

confidence: 99%

“…This fact points to hemoglobin depletion as mandatory step and so modifications to the manufacturer's protocol were included for RBC proteome analysis. Depletion of hemoglobin by cation exchange chromatography [14], by in solution pI-based fractionation [5] and by retention on a Ni(II) affinity column [15] was recently described. In the present work, we describe the use of HemogloBind TM reagent (BioTech Group) as an easy approach to deplete hemoglobin prior to 2-DE analysis of RBC (see Section 2).…”

Section: Membrane Isolation and Purification By 2-de Sample Preparatimentioning

confidence: 99%

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A novel methodology for the analysis of membrane and cytosolic sub‐proteomes of erythrocytes by 2‐DE

et al. 2009

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A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DEWith the aim of studying a wide cohort of erythrocyte samples in a clinical setting, we propose here a novel approach that allows the analysis of both human cytosolic and membrane sub-proteomes. Despite their simple structure, the high content of hemoglobin present in the red blood cells (RBCs) makes their proteome analysis enormously difficult. We investigate here different strategies for isolation of the membrane and cytosolic fractions from erythrocytes and their influence on proteome profiling by 2-DE, paying particular attention to hemoglobin removal. A simple, quick and satisfactory approach for hemoglobin depletion based on HemogloBind TM reagent was satisfactorily applied to erythrocyte cells, allowing the analysis of the cytosolic sub-proteome by 2-DE without major interference. For membrane proteome, a novel combined strategy based on hypotonic lysis isolation and further purification on minicolumns is described here, allowing detection of high molecular weight proteins (i.e. spectrin, ankyrin) and wellresolved 2-DE patterns. An aliquot of the membrane fraction was also in solution digested and analyzed by nano-LC coupled to an LTQ-Orbitrap mass spectrometer. A total of 188 unique proteins were identified by this approach. This study sets the basis for future clinical studies where the erythrocyte cell may be implicated.

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Highly Efficient Depletion Strategy for the Two Most Abundant Erythrocyte Soluble Proteins Improves Proteome Coverage Dramatically

Cited by 72 publications

References 20 publications

α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

Maximizing Peptide Identification Events in Proteomic Workflows Using Data-Dependent Acquisition (DDA)

A novel methodology for the analysis of membrane and cytosolic sub‐proteomes of erythrocytes by 2‐DE

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