Biomarker discovery in multiple myeloma by targeted absolute quantitative proteomics
The main goals of the project were i) to identify biomarkers involved in the malignant transformation from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) and ii) to establish targeted quantitative mass spectrometry at the PROMEC core facility at NTNU/HMN. To accomplish the first goal, we aimed at analyzing a cohort of 20 samples from each group (MGUS and MM). Although 30 samples from MM patients were provided by the local myeloma biobank, we faced an unpredicted challenge at obtaining a satisfactory number of MGUS samples to fully develop this part of the project. Since the last report, a few additional MGUS sample were provided by the local biobank, totalizing 10 samples. However, this number is still too small to represent the MGUS group. As this project is also part of the ongoing work of the PhD candidate Aida Demirovic, there is a continuous effort to obtain samples from MGUS patients for proper statistical validation. Hence, we are currently contacting myeloma biobanks outside Norway in an attempt to overcome this issue. We expect that by obtaining a larger number of samples from each cohort, both shotgun mass spectrometry (carried out by Aida Demirovic) and absolute targeted quantitative strategies will aid in the discovery of biomarkers within malignant transformation and potential targets for therapeutic intervention. In parallel, we have analyzed samples from a single patient at the myeloma stage and after progression to secondary plasma cell leukemia (sPCL). Here, we have employed a combination of bottoms-up proteomic profiling, targeted quantitative mass spectrometry and quantitation of epigenetically modified DNA bases. Although data collected from a case study cannot necessarily be generalized to the wider population, it can be very useful to identify potential novel drug targets relevant for high-risk disease progression. Novel potential targets will also be monitored in the MGUS and MM cohorts.
As part of this project, targeted quantitative proteome profiling was effectively implemented at NTNU in the end of 2013. Since then, the demand for this strategy, named Parallel Reaction Monitoring (PRM), has greatly increased at DMF/St.Olav as well as from outside researchers. Our group has recently published a study combining PRM analysis to show that dysregulation in uracil processing is an important factor for the development of B-cell lymphomas (Pettersen HS et al., DNA repair (2015) 25:60-71). Moreover, together with Prof. Marit Otterlei, PRM was combined with iPOND (isolation of proteins on nascent DNA) to study DNA repair proteins on newly replicated DNA and monitor their spatiotemporal orchestration at replication forks. The manuscript is in preparation and will be submitted during this spring. Furthermore, due to the advantages of PRM, especially in comparison to western blot analysis, several collaborations have been established in the last couple of years including various translational research projects. Currently, targeted mass spectrometry has been employed at NTNU to (i) investigate autophagy mechanisms in multiple myeloma (together with Prof. Geir Bjørkøy’s group); (ii) identify receptors involved in malignant transformation in MM (Prof. Anders Sundan); (iii) elucidate roles of post-translational modifications of uracil DNA glycosylase (UNG) and basic research projects within DNA repair, development and malignant transformation of lymphomas and multiple myeloma and chemotherapeutic response, as well as treatment response in colon carcinoma cells (Prof. Geir Slupphaug); (iv) several projects involving DNA repair mechanisms and neuroscience (Prof. Magnar Bjørås); (v) basic research within DNA repair and projects related to APIM therapeutics (Prof. Marit Otterlei); (vi) unravel mechanisms associated with the development of gastric cancer (Prof. Ingunn Bakke), and (vii) exercise physiology projects (Prof. Ulrik Wisløff, ISB). Thus, the establishment of targeted proteomics has brought a positive impact on several areas of medical research locally and will likely become a cornerstone technology within translational medicine at NTNU/HMN.
Targeted mass spectrometry for the identification of biomarkers involved in the progression of multiple myeloma
Targeted mass spectrometry is a highly selective approach used to detect and quantify hundreds of target proteins in a single run. As part of the present project this technique was established at the PROMEC core facility at DMF. Currently, the technique has been employed for the identification of biomarkers within malignant transformation to multiple myeloma and in collaborative scientific efforts at NTNU.
Collaboration between the Lawrence Berkeley National Laboratory (LBNL) and NTNU To become proficient on the targeted proteomics, I moved to Berkeley in September of 2012, where I spent 10 months working in the proteomics group of The Joint BioEnergy Institute (JBEI) lead by the Lawrence Berkeley National Laboratory in Berkeley, California, USA. The expertise acquired during this period was already employed in projects developed at JBEI resulting in two scientific publications (Batth TS, et al. Metabolic Engineering (2014) 26:48-56. and Weaver LJ, et al., Biotechnology and Bioengeneering (2015) 112:111-119). Targeted mass spectrometry at NTNU After returning to Trondheim, I employed the knowledge acquired in the USA to implement targeted quantitative proteome profiling at NTNU. The method, established in a Thermo QExactive instrument, is named Parallel Reaction Monitoring (PRM). In this approach, proteins of interest are quantified at peptide level, and hundreds of unique peptides representing the proteins of interest can be accurately quantified in a single run. This methodology has been increasingly applied to projects developed at NTNU. Indeed, this robust technique was recently used to investigate dysregulations in genomic uracil processing in lymphoma cell lines in our lab (published in Pettersen HS et al., DNA repair (2015) 25:60-71). In this work, 20 proteins involved in uracil processing were simultaneous quantified in B-cell lymphoma cell lines. The data provided strong evidence that persistent expression of the enzyme Activation-induced cytidine deaminase (AID) causes accumulation of genomic uracil in human B-cell malignancies. In addition, we identified targets involved in the modulation of cell metabolic pathways and oxidative stress signaling that contribute to acquired Melphalan resistance in multiple myeloma cells (Zub KA et al., PlosOne (2015), manuscript accepted). Current Status and Future Perspectives PRM is also being employed to identify of biomarkers involved in the malignant transformation from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM). In this project, uracil processing enzymes, proteins described to be dysregulated in multiple myeloma and proteins that can potentially be involved in mechanisms contributing to high-risk disease development were selected for quantification in patient samples. To determine the detectability of the selected peptides, synthetic purified peptides have been purchased and evaluated by mass spectrometry in myeloma cell lines and a small cohort of patient samples. We have now built a library of the best proteotypic peptides representing the target proteins selected for this study. Furthermore, heavy labeled isotopic versions of these peptides are now being purchased. These will be spiked in a larger cohort of patient samples as internal standards. Thus, heavy labeled peptides will serve as surrogates of the endogenous peptides enabling absolute targeted quantitative analysis of the corresponding proteins in these samples. Initially, we aimed to analyze 20 samples from each cohort (MGUS and MM). We have now a total of 20 samples from MM patients that can be included in the study. However, very few MGUS samples (2 samples) have been received so far from the biobank. We are currently working on increasing the number of MGUS samples for the absolute targeted quantitative analysis. Hopefully, the analysis of a large cohort of samples from each group will lead to the identification of biomarkers within malignant transformation that can improve diagnosis, prognosis and treatment.
Targeted mass spectrometry for the identification of biomarkers involved in the progression of multiple myeloma
Targeted mass spectrometry is a highly selective approach used to detect and quantify hundreds of protein targets in a single run. We have now established this technique at the PROMEC core facility at DMF and we aim to employ it for the identification of biomarkers within malignant transformation to multiple myeloma.
Training in the USA and return to Trondheim
To become proficient on the targeted proteomics, I spent 10 months working in the proteomics group of The Joint BioEnergy Institute (JBEI) lead by the Lawrence Berkeley National Laboratory in Berkeley, California, USA. There, I had the opportunity to learn the methodology, software and instrumentation and apply the acquired expertise in projects developed at JBEI. This stay was also scientifically fruitful as corroborated by a manuscript in preparation to be submitted to Nature Methods. After I returned to Trondheim, the mass spectrometer aimed for the present project, the ABSciex 4000 QTrap, had a major technical fault and the cost to repair it would be excessive. Nevertheless, the PROMEC had acquired a state-of-art mass spectrometer suitable for targeted peptide quantitation, the Thermo QExactive. Thus, I spent a few months learning the new instrument and using the knowledge acquired in the USA to establish targeted quantitative proteome profiling at NTNU. In the QExactive, the targeted method is known as Parallel Reaction Monitoring (PRM). In this approach, instead of selecting a few product ions in the third quadrupole in a triple quadrupole instrument, the third quadrupole is substituted with a high resolution and accurate mass (HR/AM) analyzer, permitting the parallel detection of all target product ions in one analysis.
Implementation of PRM
To implement PRM, peptides from 20 proteins involved in uracil processing were used to build a scheduled method. This was accomplished by in silico digestion of proteins of interest and selection of proteotypic peptides using the Skyline software. After selecting peptides that were unique for each protein and not likely to be modified, synthetic purified peptides were purchased and analyzed in a QExactive mass spectrometer. We have also analyzed peptides from tryptic digested recombinant proteins available in our lab. Information on retention time (RT) of these standard peptides was used to build a scheduled method which was validated in 14 cell lines, including 9 lymphoma cell lines. The method was proven to be highly sensitive and reproducible in terms of RT values and peptide abundance.
Current Status and Future Perspectives
In addition to uracil processing enzymes, proteins described to be deregulated in multiple myeloma contributing to high-risk disease development will be validated. In silico analyses of selected proteins have been performed via Skyline software and synthetic purified peptides are being purchased and evaluated by mass spectrometry. A compiled list of tested peptides will be generated and combined in a single PRM method. Myeloma cell lines and a small cohort of patient samples will be used to validate the method and determine the detectability of the selected peptides. Finally, a heavy labeled version of the best proteotypic peptides will be purchased and added to a larger cohort of patient samples as internal standards. The heavy labeled peptides will serve as surrogates of the endogenous peptides enabling absolute targeted quantitative analysis of the corresponding proteins in these samples. Prior to PRM analysis, proteins from bone marrow samples from patients will be extracted using a methanol-chloroform based method. The method was tested in several cell lines providing the highest number of proteins, as identified by mass spectrometry.
Biomarker discovery in multiple myeloma by targeted quantitative proteomics
The identification of protein biomarkers involved in the progression of multiple myeloma can potentially improve diagnosis, prognosis and treatment of the disease. Therefore we aim to discover biomarkers within malignant transformation to multiple myeloma using targeted mass spectrometry. Also, we aim to establish targeted quantitative proteome profiling at the PROMEC core facility at DMF and provide it as service to researchers in Mid-Norway.
Selected/Multiple Reaction Monitoring (MRM)
To identify clinically relevant protein biomarkers large patient- or biobank sample cohorts must be analysed. This is yet not feasible by employing traditional shotgun proteomics due to low throughput. An alternative method, named selected- or multiple reaction monitoring (SRM or MRM) can increase throughput by rather focusing upon quantitation of a preselected set of proteins.In this mass spectrometry technique specific peptides of the proteins of interest are selectively targeted for analysis in a complex sample matrix. These peptides, also called proteotypic peptides, are further fragmented in the mass spectrometer. A pair of m/z (mass to charge) ratios of the precursor (intact peptide) and a fragment ion is called a transition, and ideal transitions are used to represent the protein of interest. Notably, multiple sets of transitions can be monitored by this technology enabling the detection of multiple proteins in a single sample. The method allows rapid parallel quantitation of up to 100 proteins per sample.
To gain hands-on experience with this technology, our group has established a collaboration with the proteomics group at The Joint BioEnergy Institute (JBEI) and the Lawrence Berkeley National Laboratory (LBNL) in Berkeley, California. This group has long-standing experience in mass spectrometry and has employed S/MRM as key methodology in several projects over the last years. Thus, I moved to Berkeley in September 2012 to receive training on mass spectrometry instruments, methodologies, data analysis and softwares relevant to s/MRM at JBEI until July 2013.
Selection of target proteins
A list of over 80 proteins regulating essential processes in cancer development was generated. Most of the selected proteins, involved in processes such as B-cell immunology, disease progression, cell signaling and DNA repair, have not been previously assigned as biomarkers within malignant transformation to multiple myeloma. In addition, a few known biomarkers in multiple myeloma were included in the study as internal standards for data validation, such as for example, beta-2-microglobulin.
Selection of proteotypic peptides and mass spectrometry analysis
After the selection of candidate biomarkers, data available at peptide atlas and the Skyline software from the MacCoss lab have been employed for the selection of proteotypic peptides and their transitions. Then, synthetic purified peptides have been purchased and analyzed on a 4000QTrap mass spectrometer (ABSciex) interfaced to a nanoflow-HPLC system. The analysis of purified synthetic peptides is in progress and will continuum until a minimum of 2 proteotypic peptides per candidate protein is achieved. After that, information from retention time and best transitions will be used to verify whether the proteotypic peptides can be detected on a small cohort of patient samples. Finally, a heavy labeled version of each proteotypic peptide will be purchased and added to a larger cohort of patient samples as internal standards. The heavy labeled peptides will serve as surrogates of the endogenous peptides enabling absolute targeted quantitative analysis of the corresponding proteins in these samples.
Preparation of patient samples
To determine the best sample preparation method prior to mass spectrometry analysis, protocols for cell lysis, protein extraction and enzymatic digestion are being currently tested using cultured myeloma cells. The aim here is to find the procedure yielding the higher number of proteins, as identified by mass spectrometry. To this end, sample preparation performed according to the Filter Aided Sample Preparation (FASP) procedure, or by a modified method where a 0.1% SDS-based buffer is employed for cell lysis have demonstrated highest yield of peptides. However, the coverage of membrane proteins via these procedures is still poor. Thus, our group is currently developing an alternative method to achieve better extraction of membrane proteins in addition to intracellular proteins. After optimization, bone marrow samples from patients (CD138+) provided by the local myeloma biobank will be processed and submitted to MRM analysis.
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AID expression in B-cell lymphomas causes accumulation of genomic uracil and a distinct AID mutational signature.
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Modulation of cell metabolic pathways and oxidative stress signaling contribute to acquired melphalan resistance in multiple myeloma cells.
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