TY - JOUR
T1 - A comparison of depletion versus equalization for reducing high-abundance proteins in human serum
AU - Fernández, Carolina
AU - Santos, Hugo M.
AU - Ruíz-Romero, Cristina
AU - Blanco, Francisco J.
AU - Capelo-Martínez, José Luis
N1 - H. M. S acknowledges the post-doctoral grant SRFH/BPD/73997/2010 provided by FCT (Science and Technological Foundation). C. F. acknowledges the Foundation of the Complexo Universitario de A Coruna. wDr. J.-L. C.-M. is grateful to the Xunta de Galicia (Spain) for the program Isidro Parga Pondal and for financial support provided under project 09CSA043383PR.
PY - 2011/11/1
Y1 - 2011/11/1
N2 -
In this work three methods to diminish the content of most highly abundant proteins in human serum have been studied and compared. Protein depletion with ACN or DTT and protein equalization with the ProteoMiner
™
(PM) have been assessed by 1-D gel electrophoresis and MS. After treatment 5, 18 and 9 major proteins within the 20 most abundant proteins in serum were identified for the ACN, DTT and PM methods, respectively. The ACN method was efficient for depleting high molecular weight proteins, over 75KDa, resulting in 10±4% (n=3) of the total protein content remaining in the depleted serum. In addition, 75% of the proteins belonging to the group of the 20 most abundant proteins were not detected, making this depletion strategy a cheap alternative to expensive commercial tools regularly used for removing high abundance proteins from serum. The ACN extract was found rich in apolipoproteins. The dithithreitol method promotes the precipitation of proteins rich in disulfide bonds, mainly albumin, with 73±7% (n=3) of the total protein content remaining in the depleted serum, which was found rich in immunoglobulins. The PM method compresses the dynamic range of the serum proteins, rendering an extract containing 16±2% (n=3) of the total initial protein content. The extract was found to be rich in both apolipoproteins and immunoglobulins. As a general rule the DTT and PM methods provide a compression of the dynamic range of serum protein concentrations while the ACN method allows an effective depletion of the protein fraction above 72KDa.
AB -
In this work three methods to diminish the content of most highly abundant proteins in human serum have been studied and compared. Protein depletion with ACN or DTT and protein equalization with the ProteoMiner
™
(PM) have been assessed by 1-D gel electrophoresis and MS. After treatment 5, 18 and 9 major proteins within the 20 most abundant proteins in serum were identified for the ACN, DTT and PM methods, respectively. The ACN method was efficient for depleting high molecular weight proteins, over 75KDa, resulting in 10±4% (n=3) of the total protein content remaining in the depleted serum. In addition, 75% of the proteins belonging to the group of the 20 most abundant proteins were not detected, making this depletion strategy a cheap alternative to expensive commercial tools regularly used for removing high abundance proteins from serum. The ACN extract was found rich in apolipoproteins. The dithithreitol method promotes the precipitation of proteins rich in disulfide bonds, mainly albumin, with 73±7% (n=3) of the total protein content remaining in the depleted serum, which was found rich in immunoglobulins. The PM method compresses the dynamic range of the serum proteins, rendering an extract containing 16±2% (n=3) of the total initial protein content. The extract was found to be rich in both apolipoproteins and immunoglobulins. As a general rule the DTT and PM methods provide a compression of the dynamic range of serum protein concentrations while the ACN method allows an effective depletion of the protein fraction above 72KDa.
KW - ACN
KW - Biomarker discovery
KW - DTT
KW - Protein depletion
KW - Proteominer
UR - http://www.scopus.com/inward/record.url?scp=80055000232&partnerID=8YFLogxK
U2 - 10.1002/elps.201100183
DO - 10.1002/elps.201100183
M3 - Article
C2 - 21997478
AN - SCOPUS:80055000232
VL - 32
SP - 2966
EP - 2974
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 21
ER -