Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein

Timothy M Pawlik,Herbert Fritsche,David H Hawke,Henry M Kuerer,Kelly K Hunt,Yanna Liu,Savitri Krishnamurthy

doi:10.1186/1471-2407-6-68

Timothy M Pawlik, Herbert Fritsche + Show 5 more

Open Access

https://doi.org/10.1186/1471-2407-6-68

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Journal: BMC Cancer	Publication Date: Mar 16, 2006
Citations: 194	License type: cc-by

Affiliation: The University of Texas MD Anderson Cancer Center

Abstract

BackgroundIsotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer.MethodsPaired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed.Results353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts.ConclusionICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer.

Highlights

Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples
Using surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) MS, we previously found differences in the phenotypic proteomic profiles of nipple aspirate fluid (NAF) samples from patients with early-stage breast cancer versus healthy female volunteers [15]
We show that ICAT tandem MS is able both to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and diseasefree breasts

Summary

Introduction

Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. MS is powerful and allows, in principle, for the detection of many copurifying proteins in a fraction, it remains difficult with MS to distinguish specific from nonspecific interactions and to detect quantitative changes in protein complex abundance and composition without direct visualization of the proteins in gels [16,17,18] This is mainly because profiling experiments such as SELDI in which MS-1 only is performed, and not MS/MS, is not an inherently quantitative technique [16,19,20,21,22] and does not allow for the specific identification of individual peptides

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