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.

Assessment of Sample Preparation Bias in Mass Spectrometry-Based Proteomics

“…Various sample preparation methods have been described for bottom-up proteomics experiments targeting (solid) tissues, and a wide range of modifications to these methods can also be found in literature. 7 , 12 , 13 The most straightforward methods involve direct (in-solution) digestion of proteins without distinct procedures to remove contaminants including detergents, chaotropes, lipids, and nucleic acids. 7 , 9 , 10 In our study, we show that such an in-solution digestion (ISD) approach is a good option for quantitative proteomics featuring limited losses and good precision for peptide and protein quantification on the basis of simple and highly automatable workflows.…”

The Capacity of Long-Term In Vitro Proliferation of Acute Myeloid Leukemia Cells Supported Only by Exogenous Cytokines Is Associated with a Patient Subset with Adverse Outcome

“…The methods for analysis of proteomic and phosphoproteomic profiles in primary human AML cells have been described in detail previously [74,75,76]. Briefly, each patient sample was spiked with a heavy super-SILAC mix [77] and prepared according to the filter-aided sample preparation protocol [78] for proteome and phosphoproteome analysis using nano-liquid chromatography online coupled with a QExactive HF Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany).…”

Proteome and Phosphoproteome Changes Associated with Prognosis in Acute Myeloid Leukemia

“…We previously showed that a super-SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture) mix based on five AML cell lines provided a solid reference for quantitative proteomics studies of AML patient cells [30]. Together with optimized sample preparation and phosphopeptide enrichment protocols, our proteomics workflows proved to be useful for the study of prognosis biomarkers and treatment response in AML [31][32][33].…”