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Proteomic identification of immunoproteasome accumulation in formalin-fixed rodent spinal cords with experimental autoimmune encephalomyelitis

Jain, MR;Li, Q;Liu, T;Rinaggio, J;Ketkar, A;Tournier, V;Madura, K;Elkabes, S;Li, H;
Product: Pertussis Toxin from B. pertussis, Lyophilized in Buffer

Clinically relevant formalin-fixed and paraffin-embedded (FFPE) tissues have not been widely used in neuroproteomic studies because many proteins are presumed to be degraded during tissue preservation. Recent improvements in proteomics technologies, from the 2D gel analysis of intact proteins to the shotgun quantification of peptides and the use of isobaric tags for absolute and relative quantification (iTRAQ) method, have made the analysis of FFPE tissues possible. In recent years, iTRAQ has been one of the main methods of choice for high throughput quantitative proteomics analysis, which enables simultaneous comparison of up to eight samples in one experiment. Our objective was to assess the relative merits of iTRAQ analysis of fresh frozen versus FFPE nervous tissues by comparing experimental autoimmune encephalomyelitis (EAE)-induced proteomic changes in FFPE rat spinal cords and frozen tissues. EAE-induced proteomic changes in FFPE tissues were positively correlated with those found in the frozen tissues, albeit with ∼50% less proteome coverage. Subsequent validation of the enrichment of immunoproteasome (IP) activator 1 in EAE spinal cords led us to evaluate other proteasome and IP-specific proteins. We discovered that many IP-specific (as opposed to constitutive) proteasomal proteins were enriched in EAE rat spinal cords, and EAE-induced IP accumulation also occurred in the spinal cords of an independent mouse EAE model in a disability score-dependent manner. Therefore, we conclude that it is feasible to generate useful information from iTRAQ-based neuroproteomics analysis of archived FFPE tissues for studying neurological disease tissues.

PubMed ID: 22188123
230823082017-06-052017-06-0511:03:0111:03:012018-03-092018-03-0910:58:2510:58:25Jain, MR;Li, Q;Liu, T;Rinaggio, J;Ketkar, A;Tournier, V;Madura, K;Elkabes, S;Li, H;Jain, MR;Li, Q;Liu, T;Rinaggio, J;Ketkar, A;Tournier, V;Madura, K;Elkabes, S;Li, H;20122012Proteomic identification of immunoproteasome accumulation in formalin-fixed rodent spinal cords with experimental autoimmune encephalomyelitisProteomic identification of immunoproteasome accumulation in formalin-fixed rodent spinal cords with experimental autoimmune encephalomyelitisJournal Of Proteome ResearchJournal Of Proteome Research1791-8031791-8031111332218812322188123

Clinically relevant formalin-fixed and paraffin-embedded (FFPE) tissues have not been widely used in neuroproteomic studies because many proteins are presumed to be degraded during tissue preservation. Recent improvements in proteomics technologies, from the 2D gel analysis of intact proteins to the shotgun quantification of peptides and the use of isobaric tags for absolute and relative quantification (iTRAQ) method, have made the analysis of FFPE tissues possible. In recent years, iTRAQ has been one of the main methods of choice for high throughput quantitative proteomics analysis, which enables simultaneous comparison of up to eight samples in one experiment. Our objective was to assess the relative merits of iTRAQ analysis of fresh frozen versus FFPE nervous tissues by comparing experimental autoimmune encephalomyelitis (EAE)-induced proteomic changes in FFPE rat spinal cords and frozen tissues. EAE-induced proteomic changes in FFPE tissues were positively correlated with those found in the frozen tissues, albeit with ∼50% less proteome coverage. Subsequent validation of the enrichment of immunoproteasome (IP) activator 1 in EAE spinal cords led us to evaluate other proteasome and IP-specific proteins. We discovered that many IP-specific (as opposed to constitutive) proteasomal proteins were enriched in EAE rat spinal cords, and EAE-induced IP accumulation also occurred in the spinal cords of an independent mouse EAE model in a disability score-dependent manner. Therefore, we conclude that it is feasible to generate useful information from iTRAQ-based neuroproteomics analysis of archived FFPE tissues for studying neurological disease tissues.

Clinically relevant formalin-fixed and paraffin-embedded (FFPE) tissues have not been widely used in neuroproteomic studies because many proteins are presumed to be degraded during tissue preservation. Recent improvements in proteomics technologies, from the 2D gel analysis of intact proteins to the shotgun quantification of peptides and the use of isobaric tags for absolute and relative quantification (iTRAQ) method, have made the analysis of FFPE tissues possible. In recent years, iTRAQ has been one of the main methods of choice for high throughput quantitative proteomics analysis, which enables simultaneous comparison of up to eight samples in one experiment. Our objective was to assess the relative merits of iTRAQ analysis of fresh frozen versus FFPE nervous tissues by comparing experimental autoimmune encephalomyelitis (EAE)-induced proteomic changes in FFPE rat spinal cords and frozen tissues. EAE-induced proteomic changes in FFPE tissues were positively correlated with those found in the frozen tissues, albeit with ∼50% less proteome coverage. Subsequent validation of the enrichment of immunoproteasome (IP) activator 1 in EAE spinal cords led us to evaluate other proteasome and IP-specific proteins. We discovered that many IP-specific (as opposed to constitutive) proteasomal proteins were enriched in EAE rat spinal cords, and EAE-induced IP accumulation also occurred in the spinal cords of an independent mouse EAE model in a disability score-dependent manner. Therefore, we conclude that it is feasible to generate useful information from iTRAQ-based neuroproteomics analysis of archived FFPE tissues for studying neurological disease tissues.

5.0015.001

EAE induction, disability evaluation and tissue harvesting:

... For the mouse model, 8–10 weeks-old female C57BL/6 mice (Taconic, Hudson, NY) were immunized subcutaneously in the flank with 200 µg myelin oligodendrocyte glycoprotein peptide 35–55 (MEVGWYRSPFSRVVHLYRNGK; Stanford PAN Facility, Stanford, CA) emulsified in CFA that had been supplemented with M. tuberculosis strain H37RA (MT; Difco Laboratories, Detroit, MI) at 7 mg/ml. All mice also received an intraperitoneal injection of 350 ng pertussis toxin (List Biological, Campbell, CA) at 0 and 48 h post-immunization. ...

Author did not specify which List Labs Pertussis Toxin was utilized.  List Labs provides Product #180 - Pertussis Toxin from B. pertussis, Lyophilized in Buffer and Product #181 - Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free).

EAE induction, disability evaluation and tissue harvesting:

... For the mouse model, 8–10 weeks-old female C57BL/6 mice (Taconic, Hudson, NY) were immunized subcutaneously in the flank with 200 µg myelin oligodendrocyte glycoprotein peptide 35–55 (MEVGWYRSPFSRVVHLYRNGK; Stanford PAN Facility, Stanford, CA) emulsified in CFA that had been supplemented with M. tuberculosis strain H37RA (MT; Difco Laboratories, Detroit, MI) at 7 mg/ml. All mice also received an intraperitoneal injection of 350 ng pertussis toxin (List Biological, Campbell, CA) at 0 and 48 h post-immunization. ...

Author did not specify which List Labs Pertussis Toxin was utilized.  List Labs provides Product #180 - Pertussis Toxin from B. pertussis, Lyophilized in Buffer and Product #181 - Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free).

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312875/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312875/2012-03-022012-03-0210.1021/pr201043u10.1021/pr201043uPertussis Toxin from B. pertussis, Lyophilized in BufferPertussis Toxin from B. pertussis, Lyophilized in Bufferliho2@umdnj.eduliho2@umdnj.eduActivator;Analysis;Assess;Autoimmune;Biological;Dependent;Disease;EAE;Encephalomyelitis;Experiment;Experimental;Experimental Autoimmune Encephalomyelitis;Gel;High throughput;Identification;Injection;Intact;List;List Biological;Mouse;Neurological;Pertussis;Quantitative;Rat;Specific;Spinal;Studies;Tissue;Toxin;Journal Of Proteome ResearchActivator;Analysis;Assess;Autoimmune;Biological;Dependent;Disease;EAE;Encephalomyelitis;Experiment;Experimental;Experimental Autoimmune Encephalomyelitis;Gel;High throughput;Identification;Injection;Intact;List;List Biological;Mouse;Neurological;Pertussis;Quantitative;Rat;Specific;Spinal;Studies;Tissue;Toxin;Journal Of Proteome Research180180proteomic-identification-of-immunoproteasome-accumulationproteomic-identification-of-immunoproteasome-accumulation