title: A Method for Label-Free, Differential Top-Down Proteomics.
abstract: Biomarker discovery in the translational research has heavily relied on labeled and label-free quantitative bottom-up proteomics. Here, we describe a new approach to biomarker studies that utilizes high-throughput top-down proteomics and is the first to offer whole protein characterization and relative quantitation within the same experiment. Using yeast as a model, we report procedures for a label-free approach to quantify the relative abundance of intact proteins ranging from 0 to 30 kDa in two different states. In this chapter, we describe the integrated methodology for the large-scale profiling and quantitation of the intact proteome by liquid chromatography-mass spectrometry (LC-MS) without the need for metabolic or chemical labeling. This recent advance for quantitative top-down proteomics is best implemented with a robust and highly controlled sample preparation workflow before data acquisition on a high-resolution mass spectrometer, and the application of a hierarchical linear statistical model to account for the multiple levels of variance contained in quantitative proteomic comparisons of samples for basic and clinical research.
authors: Department of Chemistry, Northwestern University, Evanston, IL, USA.|Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA.|Proteomics Center of Excellence, Evanston, IL, USA.
authors: Ioanna
authors: I
authors: Ntai
authors: Department of Chemistry, Northwestern University, Evanston, IL, USA.|Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA.|Proteomics Center of Excellence, Evanston, IL, USA.
authors: Timothy K
authors: TK
authors: Toby
authors: Department of Chemistry, Northwestern University, Evanston, IL, USA.|Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA.|Proteomics Center of Excellence, Evanston, IL, USA.
authors: Richard D
authors: RD
authors: LeDuc
authors: Department of Chemistry, Northwestern University, Evanston, IL, USA. n-kelleher@northwestern.edu.|Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA. n-kelleher@northwestern.edu.|Proteomics Center of Excellence, Evanston, IL, USA. n-kelleher@northwestern.edu.
authors: Neil L
authors: NL
authors: Kelleher
mesh_terms: D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*
chemical_list: D020543:Proteome
keywords: Differential expression; Label-free quantitation; Proteoform; Quantitative mass spectrometry; Top-down proteomics; Top-down quantitation