TY - JOUR
T1 - Frozen tissue can provide reproducible proteomic results of subcellular fractionation
AU - Lim, Jihyeon
AU - Menon, Vilas
AU - Bitzer, Markus
AU - Miller, Leah M.
AU - Madrid-Aliste, Carlos
AU - Weiss, Louis M.
AU - Fiser, Andras
AU - Angeletti, Ruth H.
N1 - Funding Information:
This work was supported by the National Institute on Drug Abuse ( P20DA026149-03 ), the National Institute of Allergy and Infectious Diseases ( R01AI31788 ), and the National Institute of Diabetes and Digestive and Kidney Diseases ( P01DK041918-19 ).
PY - 2011/11/1
Y1 - 2011/11/1
N2 - Differential detergent fractionation (DDF) is frequently used to partition fresh cells and tissues into distinct compartments. We have tested whether DDF can reproducibly extract and fractionate cellular protein components from frozen tissues. Frozen kidneys were sequentially extracted with three different buffer systems. Analysis of the three fractions with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1693 proteins, some of which were common to all fractions and others of which were unique to specific fractions. Normalized spectral index (SIN) values obtained from these data were compared to evaluate both the reproducibility of the method and the efficiency of enrichment. SIN values between replicate fractions demonstrated a high correlation, confirming the reproducibility of the method. Correlation coefficients across the three fractions were significantly lower than those for the replicates, supporting the capability of DDF to differentially fractionate proteins into separate compartments. Subcellular annotation of the proteins identified in each fraction demonstrated a significant enrichment of cytoplasmic, cell membrane, and nuclear proteins in the three respective buffer system fractions. We conclude that DDF can be applied to frozen tissue to generate reproducible proteome coverage discriminating subcellular compartments. This demonstrates the feasibility of analyzing cellular compartment-specific proteins in archived tissue samples with the simple DDF method.
AB - Differential detergent fractionation (DDF) is frequently used to partition fresh cells and tissues into distinct compartments. We have tested whether DDF can reproducibly extract and fractionate cellular protein components from frozen tissues. Frozen kidneys were sequentially extracted with three different buffer systems. Analysis of the three fractions with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1693 proteins, some of which were common to all fractions and others of which were unique to specific fractions. Normalized spectral index (SIN) values obtained from these data were compared to evaluate both the reproducibility of the method and the efficiency of enrichment. SIN values between replicate fractions demonstrated a high correlation, confirming the reproducibility of the method. Correlation coefficients across the three fractions were significantly lower than those for the replicates, supporting the capability of DDF to differentially fractionate proteins into separate compartments. Subcellular annotation of the proteins identified in each fraction demonstrated a significant enrichment of cytoplasmic, cell membrane, and nuclear proteins in the three respective buffer system fractions. We conclude that DDF can be applied to frozen tissue to generate reproducible proteome coverage discriminating subcellular compartments. This demonstrates the feasibility of analyzing cellular compartment-specific proteins in archived tissue samples with the simple DDF method.
KW - Differential detergent fractionation
KW - Frozen tissue
KW - Normalized spectral index
KW - Subcellular location
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U2 - 10.1016/j.ab.2011.06.045
DO - 10.1016/j.ab.2011.06.045
M3 - Article
C2 - 21802400
AN - SCOPUS:80052270290
SN - 0003-2697
VL - 418
SP - 78
EP - 84
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -