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.
Highlights
Acute myeloid leukemia (AML) is a hematopoietic disease distinguished by the rapid growth of abnormally differentiated myeloid cells in the bone marrow and peripheral blood [1,2].The proliferation of the leukemic blasts interferes with the normal hematological system and it is frequently seen as a reduced production of healthy blood cells
Two technical replicates were used in the workflow test; 2 SD and DD stand for single and double digestion, respectively, U stands for urea, G stands for guanidinium hydrochloride, MM stands for mixed mode, SCX stands for strong cation exchange, FASP stands for filter-aided sample preparation; 3 The percentage of missed cleavages is calculated taken into account peptides with one or more missed cleavages
As remarked in our comparison study [15], we found it difficult to explain the poor performance of the metal oxide affinity chromatography (MOAC) phospho-enrichment, the presence of acidic amino acids within the acute myeloid leukemia (AML) phosphopeptide sequences might explain the better specificity of the immobilized metal affinity chromatography (IMAC) approach [33]
Summary
Acute myeloid leukemia (AML) is a hematopoietic disease distinguished by the rapid growth of abnormally differentiated myeloid cells in the bone marrow and peripheral blood [1,2]. System, AML is subclassified according to cellular morphology, hematopoietic lineage as well as known translocations and mutations [5] These factors contribute to different therapy responses in patients, and render the prognostication and choice of treatments difficult [1]. Mass spectrometry (MS)-based proteomic studies on AML patient samples have been increasingly published in the past years. MS proteome and phosphoproteome analysis of AML patient samples have used the standard one-dimensional electrophoresis (1DE), 2DE and the in-solution digestion method with urea in the lysis buffer to digest AML proteins prior to MS analysis or phosphorylation enrichment [10,11,12,13,14]. We will briefly review the sample preparation procedures used in previous global MS AML studies and we will describe those used in our method testing.
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