Abstract
Acute myeloid leukemia (AML) primary cells can be isolated from peripheral blood, suspended with media containing bovine serum and cryoprotectant, and stored in liquid nitrogen before being processed for proteomic analysis by mass spectrometry (MS). The presence of bovine serum and human blood proteins in AML samples can hamper the identifications of proteins, and thereby reduce the proteome coverage of the study. Herein, we have established the effect of phosphate buffered saline (PBS) washing on AML patient samples stored in media. Although PBS washes effectively removed serum and blood contaminants, the saline wash resulted in cell burst and remarkable protein material loss. We also compared different methods to preserve the AML proteome from THP-1 and Molm-13 cell lines before MS analysis: (1) stored in media containing bovine serum and dimethyl sulfoxide (DMSO); (2) stored as dried cell pellets; and (3) stored as cell lysates in 4% sodium dodecyl sulfate (SDS). MS analysis of differently preserved AML cell samples shows that preservation with DMSO produce a high number of fragile cells that will burst during freezing and thawing. Our studies encourage the use of alternative preservation methods for future MS analysis of the AML proteome.
Highlights
Acute myeloid leukemia (AML) is an aggressive hematopoietic cancer that is characterized by limited differentiation and uncontrolled proliferation of myeloid progenitor cells [1]
Freezing medium containing 20% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) is our standard for primary AML samples destined to cell culture assays, but should be removed prior to proteomic analysis [15,17]
Primary AML cells that have been stored in liquid nitrogen and 10% DMSO seem to become too fragile for phosphate buffered saline (PBS) washes prior to sample processing for mass spectrometry (MS) analysis
Summary
Acute myeloid leukemia (AML) is an aggressive hematopoietic cancer that is characterized by limited differentiation and uncontrolled proliferation of myeloid progenitor cells [1]. There is large heterogeneity among the patients regarding karyotype and genetic abnormalities, cellular phenotype, and prognosis after anti-leukemic treatment [1]. Evaluation of cytogenetic and mutational aberrations in these malignant cells is used for prognostication and treatment selection of AML patients [2], emphasizing the importance of cell biological characterization. Within the field of MS-based proteomics, large efforts have been put into optimization of sample preparation workflows, including cell lysis and protein digestion [5,6], liquid chromatography (LC)-MS instrumentation and methodology [7,8,9], and bioinformatics tools [10,11]. Contaminants that were introduced during sample collection and preparation and the effect of long-term storage on samples may induce alteration of the proteome and/or influence the MS-based protein quantification [5,14]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call