title: QuantFusion: Novel Unified Methodology for Enhanced Coverage and Precision in Quantifying Global Proteomic Changes in Whole Tissues.
abstract: Single quantitative platforms such as label-based or label-free quantitation (LFQ) present compromises in accuracy, precision, protein sequence coverage, and speed of quantifiable proteomic measurements. To maximize the quantitative precision and the number of quantifiable proteins or the quantifiable coverage of tissue proteomes, we have developed a unified approach, termed QuantFusion, that combines the quantitative ratios of all peptides measured by both LFQ and label-based methodologies. Here, we demonstrate the use of QuantFusion in determining the proteins differentially expressed in a pair of patient-derived tumor xenografts (PDXs) representing two major breast cancer (BC) subtypes, basal and luminal. Label-based in-spectra quantitative peptides derived from amino acid-coded tagging (AACT, also known as SILAC) of a non-malignant mammary cell line were uniformly added to each xenograft with a constant predefined ratio, from which Ratio-of-Ratio estimates were obtained for the label-free peptides paired with AACT peptides in each PDX tumor. A mixed model statistical analysis was used to determine global differential protein expression by combining complementary quantifiable peptide ratios measured by LFQ and Ratio-of-Ratios, respectively. With minimum number of replicates required for obtaining the statistically significant ratios, QuantFusion uses the distinct mechanisms to "rescue" the missing data inherent to both LFQ and label-based quantitation. Combined quantifiable peptide data from both quantitative schemes increased the overall number of peptide level measurements and protein level estimates. In our analysis of the PDX tumor proteomes, QuantFusion increased the number of distinct peptide ratios by 65%, representing differentially expressed proteins between the BC subtypes. This quantifiable coverage improvement, in turn, not only increased the number of measurable protein fold-changes by 8% but also increased the average precision of quantitative estimates by 181% so that some BC subtypically expressed proteins were rescued by QuantFusion. Thus, incorporating data from multiple quantitative approaches while accounting for measurement variability at both the peptide and global protein levels make QuantFusion unique for obtaining increased coverage and quantitative precision for tissue proteomes.
authors.affiliation: From the ‡Department of Biochemistry and Biophysics, §Lineberger Comprehensive Cancer Center, and.
authors.forename: Harsha P
authors.initials: HP
authors.lastname: Gunawardena
authors.affiliation: ¶Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599;
authors.forename: Jonathon
authors.initials: J
authors.lastname: O'Brien
authors.affiliation: From the ‡Department of Biochemistry and Biophysics, §Lineberger Comprehensive Cancer Center, and.
authors.forename: John A
authors.initials: JA
authors.lastname: Wrobel
authors.affiliation: From the ‡Department of Biochemistry and Biophysics, §Lineberger Comprehensive Cancer Center, and.
authors.forename: Ling
authors.initials: L
authors.lastname: Xie
authors.affiliation: ‖Division of Oncology, Washington University, St. Louis, Missouri 63110;
authors.forename: Sherri R
authors.initials: SR
authors.lastname: Davies
authors.affiliation: ‖Division of Oncology, Washington University, St. Louis, Missouri 63110;
authors.forename: Shunqiang
authors.initials: S
authors.lastname: Li
authors.affiliation: **Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Ellis
authors.affiliation: ¶Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599;
authors.forename: Bahjat F
authors.initials: BF
authors.lastname: Qaqish
authors.affiliation: From the ‡Department of Biochemistry and Biophysics, §Lineberger Comprehensive Cancer Center, and xianc@email.unc.edu.
authors.forename: Xian
authors.initials: X
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics*; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D023041:Xenograft Model Antitumor Assays
chemical_list: D000596:Amino Acids; D010455:Peptides