TY - JOUR
T1 - The influence of delay in mononuclear cell isolation on acute myeloid leukemia phosphorylation profiles
AU - van Alphen, Carolien
AU - Cucchi, David G.J.
AU - Cloos, Jacqueline
AU - Schelfhorst, Tim
AU - Henneman, Alexander A.
AU - Piersma, Sander R.
AU - Pham, Thang V.
AU - Knol, Jaco C.
AU - Jimenez, Connie R.
AU - Janssen, Jeroen J.W.M.
N1 - Funding Information: Cancer Center Amsterdam is acknowledged for support of CvA. Cancer Center Amsterdam and Netherlands Organization for Scientific Research ( NWO Middelgroot , # 91116017 ) are acknowledged for support of the mass spectrometry infrastructure. Publisher Copyright: © 2021 The Authors Copyright: Copyright 2021 Elsevier B.V., All rights reserved.
PY - 2021/4/30
Y1 - 2021/4/30
N2 - Mass-spectrometry (MS) based phosphoproteomics is increasingly used to explore aberrant cellular signaling and kinase driver activity, aiming to improve kinase inhibitor (KI) treatment selection in malignancies. Phosphorylation is a dynamic, highly regulated post-translational modification that may be affected by variation in pre-analytical sample handling, hampering the translational value of phosphoproteomics-based analyses. Here, we investigate the effect of delay in mononuclear cell isolation on acute myeloid leukemia (AML) phosphorylation profiles. We performed MS on immuno-precipitated phosphotyrosine (pY)-containing peptides isolated from AML samples after seven pre-defined delays before sample processing (direct processing, thirty minutes, one hour, two hours, three hours, four hours and 24 h delay). Up to four hours, pY phosphoproteomics profiles show limited variation. However, in samples processed with a delay of 24 h, we observed significant change in these phosphorylation profiles, with differential phosphorylation of 22 pY phosphopeptides (p < 0.01). This includes increased phosphorylation of pY phosphopeptides of JNK and p38 kinases indicative of stress response activation. Based on these results, we conclude that processing of AML samples should be standardized at all times and should occur within four hours after sample collection. Significance: Our study provides a practical time-frame in which fresh peripheral blood samples from acute myeloid patients should be processed for phosphoproteomics, in order to warrant correct interpretation of in vivo biology. We show that up to four hours of delayed processing after sample collection, pY phosphoproteomic profiles remain stable. Extended delays are associated with perturbation of phosphorylation profiles. After a delay of 24 h, JNK activation loop phosphorylation is markedly increased and may serve as a biomarker for delayed processing. These findings are relevant for biomedical acute myeloid leukemia research, as phosphoproteomic techniques are of particular interest to investigate aberrant kinase signaling in relation to disease emergence and kinase inhibitor response. With these data, we aim to contribute to reproducible research with meaningful outcomes.
AB - Mass-spectrometry (MS) based phosphoproteomics is increasingly used to explore aberrant cellular signaling and kinase driver activity, aiming to improve kinase inhibitor (KI) treatment selection in malignancies. Phosphorylation is a dynamic, highly regulated post-translational modification that may be affected by variation in pre-analytical sample handling, hampering the translational value of phosphoproteomics-based analyses. Here, we investigate the effect of delay in mononuclear cell isolation on acute myeloid leukemia (AML) phosphorylation profiles. We performed MS on immuno-precipitated phosphotyrosine (pY)-containing peptides isolated from AML samples after seven pre-defined delays before sample processing (direct processing, thirty minutes, one hour, two hours, three hours, four hours and 24 h delay). Up to four hours, pY phosphoproteomics profiles show limited variation. However, in samples processed with a delay of 24 h, we observed significant change in these phosphorylation profiles, with differential phosphorylation of 22 pY phosphopeptides (p < 0.01). This includes increased phosphorylation of pY phosphopeptides of JNK and p38 kinases indicative of stress response activation. Based on these results, we conclude that processing of AML samples should be standardized at all times and should occur within four hours after sample collection. Significance: Our study provides a practical time-frame in which fresh peripheral blood samples from acute myeloid patients should be processed for phosphoproteomics, in order to warrant correct interpretation of in vivo biology. We show that up to four hours of delayed processing after sample collection, pY phosphoproteomic profiles remain stable. Extended delays are associated with perturbation of phosphorylation profiles. After a delay of 24 h, JNK activation loop phosphorylation is markedly increased and may serve as a biomarker for delayed processing. These findings are relevant for biomedical acute myeloid leukemia research, as phosphoproteomic techniques are of particular interest to investigate aberrant kinase signaling in relation to disease emergence and kinase inhibitor response. With these data, we aim to contribute to reproducible research with meaningful outcomes.
KW - AML
KW - Acute myeloid leukemia
KW - INKA
KW - Ischemia
KW - Oxidative stress
KW - Phosphoproteomics
KW - Phosphotyrosine
KW - Preanalytical variables
KW - Proteomics
KW - Tyrosine kinase
UR - http://www.scopus.com/inward/record.url?scp=85101365318&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.jprot.2021.104134
DO - https://doi.org/10.1016/j.jprot.2021.104134
M3 - Article
C2 - 33561558
SN - 1874-3919
VL - 238
JO - Journal of Proteomics
JF - Journal of Proteomics
M1 - 104134
ER -