Extraction methods of red blood cell membrane proteins for Multidimensional Protein Identification Technology (MudPIT) analysis

Palma, Antonella De; Roveri, Antonella; Zaccarin, Mattia; Benazzi, Louise; Daminelli, Simone; Pantano, Giorgia; Buttarello, Mauro; Ursini, Fulvio; Gion, Massimo; Mauri, Pierluigi

doi:10.1016/j.chroma.2010.06.045

Cited by 26 publications

(23 citation statements)

References 37 publications

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“…Dysferlin has been well studied because of its involvement in forms of muscular dystrophy but it has never been reported to be present in the RBC membrane in previous proteome analyses. [24], [25], [26], [27] Consistent with these earlier results, we did not detect dysferlin in any of the control donor RBC membranes. We used confocal microscopy to evaluate the location of dysferlin in the RBCs of DBA patients.…”

Section: Resultssupporting

confidence: 92%

Dysferlin and Other Non-Red Cell Proteins Accumulate in the Red Cell Membrane of Diamond-Blackfan Anemia Patients

et al. 2014

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Diamond Blackfan Anemia (DBA) is a congenital anemia usually caused by diverse mutations in ribosomal proteins. Although the genetics of DBA are well characterized, the mechanisms that lead to macrocytic anemia remain unclear. We systematically analyzed the proteomes of red blood cell membranes from multiple DBA patients to determine whether abnormalities in protein translation or erythropoiesis contribute to the observed macrocytosis or alterations in the mature red blood cell membrane. In depth proteome analysis of red cell membranes enabled highly reproducible identification and quantitative comparisons of 1100 or more proteins. These comparisons revealed clear differences between red cell membrane proteomes in DBA patients and healthy controls that were consistent across DBA patients with different ribosomal gene mutations. Proteins exhibiting changes in abundance included those known to be increased in DBA such as fetal hemoglobin and a number of proteins not normally found in mature red cell membranes, including proteins involved in the major histocompatibility complex class I pathway. Most striking was the presence of dysferlin in the red blood cell membranes of DBA patients but absent in healthy controls. Immunoblot validation using red cell membranes isolated from additional DBA patients and healthy controls confirmed a distinct membrane protein signature specific to patients with DBA.

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Section: Resultssupporting

confidence: 92%

Dysferlin and Other Non-Red Cell Proteins Accumulate in the Red Cell Membrane of Diamond-Blackfan Anemia Patients

et al. 2014

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“…The one exception is a proteomics study by De Palma and co-workers that employed Triton extraction. However, that study used a low concentration of Triton-X-100 on membranes where the surface had been “shaved” using trypsin [35], which most likely affected the composition of the resulting membrane skeletons. Interestingly, they reported several unusual proteins associated with the erythrocyte membrane cytoskeleton, including subunits of chaperones containing T Complex Protein 1(TCP1).…”

Section: Introductionmentioning

confidence: 99%

Proteome analysis of the triton-insoluble erythrocyte membrane skeleton

Basu

Harper

Pesciotta

et al. 2015

Journal of Proteomics

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Erythrocyte shape and membrane integrity is imparted by the membrane skeleton, which can be isolated as a Triton X-100 insoluble structure that retains the biconcave shape of intact erythrocytes, indicating isolation of essentially intact membrane skeletons. These erythrocyte “Triton Skeletons” have been studied morphologically and biochemically, but unbiased proteome analysis of this substructure of the membrane has not been reported. In this study, different extraction buffers and in-depth proteome analyses were used to more fully define the protein composition of this functionally critical macromolecular complex. As expected, the major, well-characterized membrane skeleton proteins and their associated membrane anchors were recovered in good yield. But surprisingly, a substantial number of additional proteins that are not considered in erythrocyte membrane skeleton models were recovered in high yields, including myosin-9, lipid raft proteins (stomatin, flotillin1 and 2), multiple chaperone proteins (HSPs, protein disulfide isomerase and calnexin), and several other proteins. These results show the membrane skeleton is substantially more complex than previous biochemical studies indicated, and it apparently has localized regions with unique protein compositions and functions. This comprehensive catalog of the membrane skeleton should lead to new insights into erythrocyte membrane biology and pathogenic mutations that perturb membrane stability.

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“…These techniques include white ghost (WG) analysis on 1-[1] or 2-DE gels,[2] in-solution digestion of four RBC fractions (white ghosts, cytoplasmic proteins, inside out vesicles (IOV), and membrane skeletal proteins),[3] and membrane protein extraction with detergents followed by in-solution digestion for multidimensional protein identification technology analysis (MudPIT). [4] Several studies used 2-DE as the preferred fractionation method for LC-MS/MS analysis of the RBC proteome. [5–8] However, 2-DE analysis of hydrophobic membrane proteins is not ideal and detection can vary significantly depending upon individual protein structures and characteristics.…”

Section: Introductionmentioning

confidence: 99%

A label-free proteome analysis strategy for identifying quantitative changes in erythrocyte membranes induced by red cell disorders

Pesciotta

Sriswasdi

Tang

et al. 2012

Journal of Proteomics

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Red blood cells have been extensively studied but many questions regarding membrane properties and pathophysiology remain unanswered. Proteome analysis of red cell membranes is complicated by a very wide dynamic range of protein concentrations as well as the presence of proteins that are very large, very hydrophobic, or heterogeneously glycosylated. This study investigated removal of other blood cell types, red cell membrane extraction, differing degrees of fractionation using 1-D SDS gels, and label-free quantitative methods to determine optimized conditions for proteomic comparisons of clinical blood samples. The results showed that fractionation of red cell membranes on 1-D SDS gels was more efficient than low-ionic-strength extractions followed by 1-D gel fractionation. When gel lanes were sliced into 30 uniform slices, a good depth of analysis was obtained that included identification of most well-characterized, low-abundance red cell membrane proteins including those present at 500 to 10,000 copies per cell. Furthermore, the size separation enabled detection of changes due to proteolysis or in vivo protein crosslinking. A combination of Rosetta Elucidator quantitation and subsequent statistical analysis enabled the robust detection of protein differences that could be used to address unresolved questions in red cell disorders.

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Extraction methods of red blood cell membrane proteins for Multidimensional Protein Identification Technology (MudPIT) analysis

Cited by 26 publications

References 37 publications

Dysferlin and Other Non-Red Cell Proteins Accumulate in the Red Cell Membrane of Diamond-Blackfan Anemia Patients

Dysferlin and Other Non-Red Cell Proteins Accumulate in the Red Cell Membrane of Diamond-Blackfan Anemia Patients

Proteome analysis of the triton-insoluble erythrocyte membrane skeleton

A label-free proteome analysis strategy for identifying quantitative changes in erythrocyte membranes induced by red cell disorders

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