JIOMICS

JIOMICS | VOL 5 | ISSUE 2 | DECEMBER 2015 | 1-62
JOURNAL OF INTEGRATED OMICS
Journal of Integrated Omics
A METHODOLOGICAL JOURNAL
HTTP://WWW.JIOMICS.COM
Special Issue: Proceeding Abstracts of the 4 th International Congress on Analytical Proteomics (ICAP 2015)
Methods for the preparation of acute myeloid leukemia patient samples for
proteomic and phosphoproteomic analysis
M. Hernandez-Valladares* 1 , E. Aasebø 1 , F. Berven 1 , Ø. Bruserud 2 , F. Selheim 1
1
PROBE, Building for Basic Biology, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway. 2 Department of Internal Medicine,
Haukeland University Hospital, N-5020, Bergen, Norway. *Corresponding author: Maria.Hernandez-Valladares@uib.no
Available Online: 31 December 2015
Abstract
Purpose: The study of global protein expression in acute myeloid leukemia (AML) patients by mass spectrometry (MS) can help identifying
differential expression and post-translational modifications of proteins that could represent disease-related biomarkers for early diagnosis
or for improved prognostics and to predict the patient response to different therapeutics. To optimize the proteome and phosphoproteome
coverage of samples from AML patients by LC-MS/MS analysis, we have tested several methods with different peptide fractionation and phosphopeptide
enrichment strategies.
Experimental description: Peptide samples were prepared with an in-solution digestion protocol, using urea or guanidinium hydrochloride
as denaturant, and with the filter aided sample preparation (FASP) methodology. Peptide fractionation was carried out with reverse phase/
strong cation exchange (SCX) and SCX disks in a stage-tip. Techniques for phosphopeptide enrichment included metal oxide affinity chromatography
(MOAC), immobilized metal affinity chromatography (IMAC) and sequential elution from IMAC (SIMAC).
Results: From an initial screening of the different samples on a Linear Trap Quadrupole (LTQ) Orbitrap Elite MS, we found the best proteome
coverage with the sequential FASP method, with Lys-C and trypsin as digestion enzymes, which identified and quantified 3100 proteins
from 20 μg of sample. With the same strategy, followed by a separate MOAC/TiO 2 -beads enrichment of the two peptide pools, we identified
and quantified 2.900 phosphorylation sites from only 250 μg of AML patient sample. On a QExactive HF hybrid Quadrupole-Orbitrap MS,
5400 proteins and 4000 phosphosites were identified and quantified from these FASP-prepared samples.
Conclusions: To improve the proteome and phosphoproteome coverage of AML patient samples by MS analysis to discover new biomarkers,
testing of different methods can be beneficial. Based on sample preparation optimization in our laboratory, we have chosen the
FASP protocol to prepare AML patient samples for MS-based proteomic and phosphoproteomic studies.
Keywords: AML, proteomics, phosphoproteomics, sample preparation.
Acknowledgements: The authors would like to thank the Norwegian Cancer Society and Øyvinn Mølbach-Petersens Fond for the funding
of this work.
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