title: Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia.
abstract: Global mass spectrometry (MS)-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML) biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP) as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility.
authors.affiliation: Department of Biomedicine, Faculty of Medicine and Dentistry, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway. Maria.Hernandez-Valladares@uib.no.
authors.forename: Maria
authors.initials: M
authors.lastname: Hernandez-Valladares
authors.affiliation: Department of Biomedicine, Faculty of Medicine and Dentistry, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway. Elise.Aasebo@uib.no.
authors.forename: Elise
authors.initials: E
authors.lastname: Aasebø
authors.affiliation: Department of Biomedicine, Faculty of Medicine and Dentistry, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway. Frode.Selheim@uib.no.
authors.forename: Frode
authors.initials: F
authors.lastname: Selheim
authors.affiliation: Department of Biomedicine, Faculty of Medicine and Dentistry, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway. Frode.Berven@uib.no.
authors.forename: Frode S
authors.initials: FS
authors.lastname: Berven
authors.affiliation: Department of Clinical Science, Faculty of Medicine and Dentistry, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway. Oystein.Bruserud@uib.no.
authors.forename: Øystein
authors.initials: Ø
authors.lastname: Bruserud
keywords: FASP; IMAC; SILAC; StageTip; acute myeloid leukemia; biomarker; mass spectrometry; phosphoproteomics; proteomics; sample preparation