Freezing effects on the acute myeloid leukemia cell proteome and phosphoproteome revealed using optimal quantitative workflows

Aasebø, Elise; Mjaavatten, Olav; Vaudel, Marc; Farag, Yehia; Selheim, Frode; Berven, Frode S.; Bruserud, Øystein; Hernandez-Valladares, Maria

doi:10.1016/j.jprot.2016.03.049

Cited by 36 publications

(38 citation statements)

References 77 publications

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“…The use of the in-solution method with guanidinium hydrochloride, which simultaneously performs reduction and alkylation at high temperature and therefore preserves the sample integrity and its phosphorylation status, resulted in a high number of quantified proteins. However, a high percentage of missed cleavages in the peptide sequences and a poor reproducibility of the phospho-enrichment workflows using peptide samples obtained with this method discouraged us from its further use at a large scale [15]. …”

Section: Discussionmentioning

confidence: 99%

“…Therefore, we chose the IMAC strategy for the enrichment of phosphopeptides prepared with the FASP/trypsin procedure on AML patient samples. As remarked in our comparison study [15], we found it difficult to explain the poor performance of the MOAC (and consequentially that of the SIMAC) phospho-enrichment, although the presence of acidic amino acids within the AML phosphopeptide sequences might explain the better specificity of the IMAC approach [33]. …”

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

confidence: 98%

“…In order to find out the optimal quantitative workflows for MS studies of AML patient samples, we have recently tested most of the sample preparation methods described above [15]. We used blast cells from peripheral blood of an AML patient for all the proteomic workflows while we used pooled samples from several AML patients for all the phospho-enrichment workflows.…”

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

confidence: 99%

“…We kept the MS acquisition time constant or comparable for all the analyses. A detailed description of the LC-MS conditions can be found in the Supplemental Methods of our comparison study [15]. …”

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

confidence: 99%

“…Published global 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 have recently tested several methods of sample preparation for the MS analysis of the AML blasts that included in-solution digestion methods with urea and guanidinium hydrochloride as denaturant reagents in the lysis buffer and filter-aided sample preparation (FASP) [15,16]. Several methods of phosphorylation enrichment, immobilized metal affinity chromatography (IMAC) [17], metal oxide affinity chromatography (MOAC) [18] and sequential elution from IMAC (SIMAC) [19] were also included in our tests.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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

confidence: 98%

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

confidence: 99%

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

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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Effect of storage time and temperature on cell cycle analysis by mass cytometry

2018

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Cell cycle analysis is a recognized and important application of flow cytometry and, more recently, mass cytometry (MCM). Both technologies have been utilized for analysis of the cell cycle state of ex vivo samples from patients with hematologic malignancies. Clinical samples are frequently stored for hours at room temperature or cryogenically frozen before processing and analysis; however, how these processing methods alter cell cycle state is not well described. To understand how storage time and temperature affect the analysis of cell cycle distribution by MCM, two leukemia cell lines, HL-60 and MOLM13, and primary human cells from three human bone marrow aspirates were stored and frozen under a variety of conditions that are likely to be encountered in a clinical setting. Our findings indicate that short delays in sample processing (less than 1 h), have little to no effect on cell cycle distribution, while longer delays or cryopreservation cause significant disruptions to the cell cycle fraction characterized by consistent reductions in IdU incorporation and variable alterations in other cell cycle phases. Analysis of the recovery of cryopreserved leukemia cell lines and marrow cells demonstrated that cell cycle alterations persist for at least 48 h after thawing. Our findings demonstrate that accurate cell cycle analysis requires that samples be processed rapidly after collection, and that cryopreservation significantly alters cell cycle fractions. Measurement of IdU incorporation was the most sensitive to both delays in processing and cryopreservation, while estimation of the total cycling cell fraction using Ki-67 or phosphorylated retinoblastoma protein were least altered by the conditions tested. These findings provide guidance for the ideal approach to collection of samples for cell cycle analysis and can aid interpretation of cell cycle data from samples that cannot be collected under ideal circumstances.

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Impact of phosphoproteomics in the translation of kinase‐targeted therapies

et al. 2016

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Signaling pathways driven by protein and lipid kinases are altered in most human diseases. Therefore, pharmacological inhibitors of cell signaling are one of the most intensively pursued therapeutic approaches for the treatment of diseases such as cancer, neurodegeneration, and metabolic syndromes. Phosphoproteomics is a technique that measures the products of kinase activities and, with the appropriate bioinformatics techniques, the methodology can also provide measures of kinase pathway activation and network circuitry. Hence, due to recent technological advantages, LC-MS-based quantitative phosphoproteomics provides relevant information for the design and implementation of kinase inhibitor based therapies. Here, we review how phosphoproteome profiling is being used in translational research as a means to identify drug targets and biomarkers for personalizing therapies based on kinase inhibitors.

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Freezing effects on the acute myeloid leukemia cell proteome and phosphoproteome revealed using optimal quantitative workflows

Cited by 36 publications

References 77 publications

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

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

Effect of storage time and temperature on cell cycle analysis by mass cytometry

Impact of phosphoproteomics in the translation of kinase‐targeted therapies

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