Performance of super‐SILAC based quantitative proteomics for comparison of different acute myeloid leukemia (AML) cell lines

Aasebø, Elise; Vaudel, Marc; Mjaavatten, Olav; Gausdal, Gro; Burgh, Arthur Van der; Gjertsen, Bjørn Tore; Døskeland, Stein Ove; Bruserud, Øystein; Berven, Frode S.; Selheim, Frode

doi:10.1002/pmic.201300448

Cited by 35 publications

(24 citation statements)

References 27 publications

(30 reference statements)

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“…Thus, there is a risk of losing quantification of proteins only present in the patient sample. However, in our hands, the percentage of proteins in AML patient samples that could not be quantified with a super-SILAC mix of 5 AML cell lines [40] never exceeded 2% (unpublished data). With isobaric labeling techniques targeting the N-terminus and side chain amines of peptides such as isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tag (TMT) it is possible to compare up to eight and ten samples, respectively.…”

Section: Ms-based Methodologies For the Study The Aml Proteomementioning

confidence: 89%

“…Their analysis of T-ALL and AML cell lines revealed the same tendencies, and showed that AML cell lines and AML patient cells did not cluster together. Hierarchical clustering of protein expression in five AML cell lines (compared and characterized in [40]) and twenty-seven AML patient samples from unpublished shotgun proteomics data from our lab showed that 560 of the 1410 proteins quantified in all thirty-two samples were significantly differently expressed in the cell lines compared to primary cells. Proteins involved in processes such as translational initiation and elongation were higher expressed in the cell lines, while proteins involved in or part of the mitochondrion were lower expressed in the cell lines, compared to the primary patient cells.…”

Section: Sample Considerationsmentioning

confidence: 99%

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Global Cell Proteome Profiling, Phospho-signaling and Quantitative Proteomics for Identification of New Biomarkers in Acute Myeloid Leukemia Patients

Aasebø¹,

Forthun²,

Berven³

et al. 2015

CPB

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The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can be quantified with high confidence in new mass spectrometers. While recent reports aiming to propose proteomic or phosphoproteomic biomarkers on the studied AML patient samples have taken advantage of the technological progress, the access to large cohorts of AML patients to sample from and the availability of appropriate control samples still remain challenging.

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Section: Ms-based Methodologies For the Study The Aml Proteomementioning

confidence: 89%

Section: Sample Considerationsmentioning

confidence: 99%

Global Cell Proteome Profiling, Phospho-signaling and Quantitative Proteomics for Identification of New Biomarkers in Acute Myeloid Leukemia Patients

Aasebø¹,

Forthun²,

Berven³

et al. 2015

CPB

Self Cite

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show abstract

“…An AML spike-in reference was generated by combining Arg6-and Lys8-labeled protein cell lysates from five heterogeneous AML-derived cell lines, according to the super-SILAC (Stable Isotope Labeling with Amino acids in Cell culture) mix approach. 29,30 The spike-in reference was added to each patient sample in an 1:2 ratio (w:w; super-SILAC mix:AML patient sample) for SILAC-based quantitation.…”

Section: Aml Super-silac MIXmentioning

confidence: 99%

Proteome and phosphoproteome changes associated with prognosis in acute myeloid leukemia

Aasebø

Berven

Bartaula-Brevik

et al. 2019

Preprint

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word count: 213 Number of figures and tables: 5 Number of references: 84 Key Points  V-ATPases and RNA processing proteins are downregulated and upregulated, respectively, in relapse patients  Relapse patients show higher activity of CSK2 and CDKs when compared to relapse-free patients AbstractAcute myeloid leukemia (AML) is a hematological cancer that mainly affects the elderly.Although complete remission (CR) is achieved for majority of the patients after induction and consolidation therapies, nearly two-thirds relapse within a short interval. Understanding biological factors that determine relapse has therefore become of major clinical interest in AML.We utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify protein changes and protein phosphorylation events associated with AML relapse in primary cells from 41 AML patients at time of diagnosis. Patients were defined as relapse-free if they had not relapsed within a 5-year clinical follow-up after AML diagnosis. Relapse was associated with increased expression of RNA processing proteins and decreased expression of V-ATPase proteins. We also observed an increase in phosphorylation events catalyzed by cyclin-dependent kinases (CDKs) and casein kinase 2 (CSK2). The biological relevance of the proteome findings was supported by cell proliferation assays using inhibitors of V-ATPase (bafilomycin), CSK2 (CX-4945), CDK4/6 (abemaciclib) and CDK2/7/9 (SNS-032). While bafilomycin preferentially inhibited the cells from relapse patients, the kinase inhibitors were less efficient in these cells.This suggests that therapy against the upregulated kinases also could target the factors inducing their upregulation rather than their activity. In conclusion, our study presents markers that could help predict AML relapse and direct therapeutic strategies.

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“…Although the tests were carried out with label-free and stable isotope labeling with amino acids in cell culture (SILAC)-labeled AML patient samples, we will only review herein our results from the SILAC-quantified samples. We used 20 µg of AML lysate and 20 µg of super-SILAC mix [30] for each proteomic workflow. We have also included two fractionation strategies using the StageTip format and the procedures with polystyrenedivinylbenzene reversed phase sulfonate (SDB-RPS, so-called mixed mode, MM) and SCX plugs described by Kulak et al [26].…”

Section: Testing Of Standard and Novel Ms-based Proteomic And Phosmentioning

confidence: 99%

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

et al. 2016

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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.

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Performance of super‐SILAC based quantitative proteomics for comparison of different acute myeloid leukemia (AML) cell lines

Cited by 35 publications

References 27 publications

Global Cell Proteome Profiling, Phospho-signaling and Quantitative Proteomics for Identification of New Biomarkers in Acute Myeloid Leukemia Patients

Global Cell Proteome Profiling, Phospho-signaling and Quantitative Proteomics for Identification of New Biomarkers in Acute Myeloid Leukemia Patients

Proteome and phosphoproteome changes associated with prognosis in acute myeloid leukemia

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

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