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Proteomes 2016, 4(3), 24; doi:10.3390/proteomes4030024

Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

1
Department of Biomedicine, Faculty of Medicine and Dentistry, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway
2
Department of Clinical Science, Faculty of Medicine and Dentistry, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway
*
Author to whom correspondence should be addressed.
Academic Editors: Edwin Lasonder and Jacek R. Wisniewski
Received: 6 July 2016 / Revised: 9 August 2016 / Accepted: 12 August 2016 / Published: 22 August 2016
(This article belongs to the Special Issue Clinical Proteomics)
View Full-Text   |   Download PDF [527 KB, uploaded 22 August 2016]   |  

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. View Full-Text
Keywords: acute myeloid leukemia; proteomics; phosphoproteomics; sample preparation; FASP; SILAC; mass spectrometry; IMAC; StageTip; biomarker acute myeloid leukemia; proteomics; phosphoproteomics; sample preparation; FASP; SILAC; mass spectrometry; IMAC; StageTip; biomarker
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MDPI and ACS Style

Hernandez-Valladares, M.; Aasebø, E.; Selheim, F.; Berven, F.S.; Bruserud, Ø. Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia. Proteomes 2016, 4, 24.

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