title: Missing Value Monitoring Enhances the Robustness in Proteomics Quantitation.
abstract: In global proteomic analysis, it is estimated that proteins span from millions to less than 100 copies per cell. The challenge of protein quantitation by classic shotgun proteomic techniques relies on the presence of missing values in peptides belonging to low-abundance proteins that lowers intraruns reproducibility affecting postdata statistical analysis. Here, we present a new analytical workflow MvM (missing value monitoring) able to recover quantitation of missing values generated by shotgun analysis. In particular, we used confident data-dependent acquisition (DDA) quantitation only for proteins measured in all the runs, while we filled the missing values with data-independent acquisition analysis using the library previously generated in DDA. We analyzed cell cycle regulated proteins, as they are low abundance proteins with highly dynamic expression levels. Indeed, we found that cell cycle related proteins are the major components of the missing values-rich proteome. Using the MvM workflow, we doubled the number of robustly quantified cell cycle related proteins, and we reduced the number of missing values achieving robust quantitation for proteins over ∼50 molecules per cell. MvM allows lower quantification variance among replicates for low abundance proteins with respect to DDA analysis, which demonstrates the potential of this novel workflow to measure low abundance, dynamically regulated proteins.
authors.affiliation: IFOM, the FIRC Institute of Molecular Oncology, Milan 20139, Italy.
authors.forename: Vittoria
authors.initials: V
authors.lastname: Matafora
authors.affiliation: IFOM, the FIRC Institute of Molecular Oncology, Milan 20139, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Corno
authors.affiliation: IFOM, the FIRC Institute of Molecular Oncology, Milan 20139, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Ciliberto
authors.affiliation: IFOM, the FIRC Institute of Molecular Oncology, Milan 20139, Italy.
authors.forename: Angela
authors.identifier: 0000-0003-4842-6556
authors.initials: A
authors.lastname: Bachi
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification*; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification*; D020543:Proteome / Q000235:genetics*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D018797:Cell Cycle Proteins; D010455:Peptides; D020543:Proteome
keywords: cell cycle; data-dependent acquisition (DDA); data-independent acquisition (DIA); mass spectrometry; missing value monitoring (MvM) workflow; missing values; proteomics