Multilectin affinity chromatography for characterization of multiple glycoprotein biomarker candidates in serum from breast cancer patients.
BACKGROUND
Glycoproteins are often associated with cancer and are important in serum studies, for which glycosylation is a common posttranslational modification.


METHODS
We used multilectin affinity chromatography (M-LAC) to isolate glycoproteins from the sera of breast cancer patients and controls. The proteins were identified by HPLC-tandem mass spectrometry (MS/MS) analysis of the corresponding tryptic digests. We used the FuncAssociate Gene Ontology program for association analysis of the identified proteins. Biomarker candidates in these groups were comparatively quantitated by use of peak area measurements, with inclusion of an internal standard. We analyzed data for concordance within the ontology association groups for vector of change with the development of breast cancer.


RESULTS
Detection of the known low-concentration biomarker HER-2 (8-24 mug/L) enabled us to establish a dynamic range of 10(6), relative to the amount of albumin, for the depletion step. We then used ELISA to confirm this range. Proteins associated with lipid transport and metabolism, cell growth and maintenance, ion homeostasis, and protease inhibition were found to be differentially regulated in serum from women with breast cancer compared with serum from women without breast cancer.


CONCLUSIONS
M-LAC for isolation of the serum glycoproteome, coupled with liquid chromatography-MS/MS and the use of gene ontology associations, can be used to characterize large panels of candidate markers, which can then be evaluated in a particular patient population.
Yang
Ziping
Z
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
Harris
Lyndsay E
LE
Palmer-Toy
Darryl E
DE
Hancock
William S
WS
D014408:Biomarkers, Tumor / Q000097:blood*; D001943:Breast Neoplasms / Q000175:diagnosis*; D018270:Carcinoma, Ductal, Breast / Q000175:diagnosis*; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000175:diagnosis*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D037102:Lectins; D020543:Proteome / Q000032:analysis*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000097:blood; D044967:Serum
D014408:Biomarkers, Tumor; D006023:Glycoproteins; D037102:Lectins; D020543:Proteome; D018719:Erb-b2 Receptor Tyrosine Kinases