Elise Aasebø

Discovery and initial verification of differentially abundant proteins between multiple sclerosis patients and controls using iTRAQ and SID-SRM.

In the present study, we aimed to discover cerebrospinal fluid (CSF) proteins with significant abundance difference between early multiple sclerosis patients and controls, and do an initial verification of these proteins using selected reaction monitoring (SRM). iTRAQ and Orbitrap MS were used to compare the CSF proteome of patients with clinically isolated syndrome (CIS) (n=5), patients with relapsing-remitting multiple sclerosis that had CIS at the time of lumbar puncture (n=5), and controls with other inflammatory neurological disease (n=5). Of more than 1200 identified proteins, five proteins were identified with significant abundance difference between the patients and controls. In the initial verification using SRM we analyzed a larger patient and control cohort (n=132) and also included proteins reported as differentially abundant in multiple sclerosis in the literature. We found significant abundance difference for 11 proteins after verification, of which the five proteins alpha-1-antichymotrypsin, contactin-1, apolipoprotein D, clusterin, and kallikrein-6 were significantly differentially abundant in several of the group comparisons. This initial study form the basis for further biomarker verification studies in even larger sample cohorts, to determine if these proteins have relevance as diagnostic or prognostic biomarkers for multiple sclerosis.

Effects of blood contamination and the rostro-caudal gradient on the human cerebrospinal fluid proteome.

Over the last years there has been an increased focus on the importance of knowing the effect of pre-analytical influence on the proteomes under study, particularly in the field of biomarker discovery. We present three proteomics studies examining the effect of blood contamination and the rostro-caudal gradient (RCG) on the cerebrospinal fluid (CSF) proteome, in addition to plasma/CSF protein ratios. The studies showed that the central nervous system (CNS) derived proteins appeared to be unaffected by the RCG, while the plasma-derived proteins showed an increase in concentration towards the lumbar area. This implies that the concentration of the plasma-derived proteins in CSF will vary depending on the volume of CSF that is collected. In the CSF samples spiked with blood, 262 of 814 quantified proteins showed an abundance increase of more than 1.5 fold, while 403 proteins had a fold change of less than 1.2 and appeared to be unaffected by blood contamination. Proteins with a high plasma/CSF ratio appeared to give the largest effect on the CSF proteome upon blood contamination. The results give important background information on how factors like blood contamination, RCG and blood-CNS-barrier influences the CSF proteome. This information is particularly important in the field of biomarker discovery, but also for routine clinical measurements. The data from the blood contamination and RCG discovery studies have been deposited to the ProteomeXchange with identifier PXD000401.

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

As a direct consequence of the high diversity of the aggressive blood cancer acute myeloid leukemia (AML), proteomic samples from patients are strongly heterogeneous, rendering their accurate relative quantification challenging. In the present study, we investigated the benefits of using a super‐SILAC mix of AML derived cell lines as internal standard (IS) for quantitative shotgun studies. The Molm‐13, NB4, MV4‐11, THP‐1, and OCI‐AML3 cell lines were selected for their complementarity with regard to clinical, cytogenetic, and molecular risk factors used for prognostication of AML patients. The resulting IS presents a high coverage of the AML proteome compared to single cell lines allied with high technical reproducibility, thus enabling its use for AML patient comparison. This was confirmed by comparing the protein regulation between the five cell lines and by applying the IS to patient material; hence, we were able to reproduce specific functional regulations known to be related to disease progression and molecular genetic abnormalities. The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000441.

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

UNLABELLED MS-based proteomic studies aiming for the discovery of acute myeloid leukemia (AML) biomarkers require sample processing that can assure an optimal proteome coverage and identification of PTMs. We evaluated different in-solution and filter-aided sample preparation (FASP) proteomic workflows and different enrichment strategies of phosphorylated peptides. The FASP protocols in the label-free and SILAC (stable isotope labelling with amino acids in cell culture) approaches were selected for producing the highest number of quantified proteins with reduced number of missed cleavages. The IMAC method was selected for producing the highest number of quantified phosphopeptides from SILAC-labelled peptides prepared with FASP. Using these selected workflows, we studied the effect of liquid nitrogen storage on the proteome and phosphoproteome of four AML patients. Our results showed that although there was not a major global proteome and phosphoproteome change when compared to their freshly processed counterparts, the freezing appeared to influence the abundance of mitochondrial proteins involved in the respiratory chain transport and affect the phosphorylation of apoptosis related proteins, cell surface interactors, ERK/MAPK targets and proteins involved in thrombin signalling. Our results encourage the assessment of current procedures of AML sample collection and preservation that could be used in future AML biomarker discovery studies. BIOLOGICAL SIGNIFICANCE Proteomic studies aiming to identify potential cancer biomarkers need to utilize the best sample preparation workflows on the samples of interest to achieve maximal proteome coverage. We have tested the most popular and recent proteomic and phosphoproteomic methods on cell lysates from patients with AML and systematically evaluated their performance. Our study shows the relevance of selecting the patient sample procedure giving the highest protein and PTM coverage. Moreover, we assessed how the proteome and phosphoproteome were affected by the conventional liquid nitrogen storage compared to cell lysis of fresh material, using the methods that worked best in our hands. For potential biomarkers that could be used for AML diagnostic and prognostic, it is of great importance to study the behaviour during sample conservation in order to avoid artefactual findings. Our results recommend caution in data interpretation when using different protocols of sample collection and conservation for proteomic and phosphosproteomic research.

Quantitative proteomics suggests decrease in the secretogranin-1 cerebrospinal fluid levels during the disease course of multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS with unknown cause. Proteins with different abundance in the cerebrospinal fluid (CSF) from relapsing‐remitting MS (RRMS) patients and neurological controls could give novel insight to the MS pathogenesis and be used to improve diagnosis, predict prognosis and disease course, and guide in therapy decisions. We combined iTRAQ labeling and Orbitrap mass spectrometry to discover proteins with different CSF abundance between six RRMS patients and 18 neurological disease controls. From 777 quantified proteins seven were selected as biomarker candidates, namely chitinase‐3‐like protein 1, secretogranin‐1 (Sg1), cerebellin‐1, neuroserpin, cell surface glycoprotein MUC18, testican‐2 and glutamate receptor 4. An independent sample set of 13 early‐MS patients, 13 RRMS patients and 13 neurological controls was used in a multiple reaction monitoring verification study. We found the intracellular calcium binding protein Sg1 to be increased in early‐MS patients compared to RRMS and neurological controls. Sg1 should be included in further studies to elucidate its role in the early phases of MS pathogenesis and its potential as a biomarker for this disease.

Label free analysis of human cerebrospinal fluid addressing various normalization strategies and revealing protein groups affected by multiple sclerosis.

The aims of the study were to: (i) identify differentially regulated proteins in cerebrospinal fluid (CSF) between multiple sclerosis (MS) patients and non‐MS controls; (ii) examine the effect of matching the CSF samples on either total protein amount or volume, and compare four protein normalization strategies for CSF protein quantification. CSF from MS patients (n = 37) and controls (n = 64), consisting of other noninflammatory neurological diseases (n = 50) and non neurological spinal anesthetic subjects (n = 14), were analyzed using label‐free proteomics, quantifying almost 800 proteins. In total, 122 proteins were significantly regulated (p < 0.05), where 77 proteins had p‐value <0.01 or AUC value >0.75. Hierarchical clustering indicated that there were two main groups of MS patients, those with increased levels of inflammatory response proteins and decreased levels of proteins involved in neuronal tissue development (n = 30), and those with normal protein levels for both of these protein groups (n = 7). The main subgroup of controls clustering with the MS patients showing increased inflammation and decreased neuronal tissue development were patients suffering from chronic fatigue. Our data indicate that the preferable way to quantify proteins in CSF is to first match the samples on total protein amount and then normalize the data based on the median intensities, preferably from the CNS‐enriched proteins.

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

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.

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

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.

Therapeutic targeting of leukemic stem cells in acute myeloid leukemia – the biological background for possible strategies

ABSTRACT Introduction: Acute myeloid leukemia (AML) is an aggressive malignancy, caused by the accumulation of immature leukemic blasts in blood and bone marrow. There is a relatively high risk of chemoresistant relapse even for the younger patients who can receive the most intensive antileukemic treatment. Treatment directed against the remaining leukemic and preleukemic stem cells will most likely reduce the risk of later relapse. Areas covered: Relevant publications were identified through literature searches. The authors searched for original articles and recent reviews describing (i) the characteristics of leukemic/preleukemic stem cells; (ii) the importance of the bone marrow stem cell niches in leukemogenesis; and (iii) possible therapeutic strategies to target the preleukemic/leukemic stem cells. Expert opinion: Leukemia relapse/progression seems to be derived from residual chemoresistant leukemic or preleukemic stem cells, and a more effective treatment directed against these cells will likely be important to improve survival both for patients receiving intensive treatment and leukemia-stabilizing therapy. Several possible strategies are now considered, including the targeting of the epigenetic regulation of gene expression, proapoptotic intracellular signaling, cell metabolism, telomere activity and the AML-supporting effects by neighboring stromal cells. Due to disease heterogeneity, the most effective stem cell-directed therapy will probably differ between individual patients.

Rethinking the role of osteopontin in human acute myeloid leukemia.

Osteopontin is a secreted glycoprotein that binds to specific receptors and influences the functions of a wide range of cells. It acts both as chemoattractant cytokine and as an extracellular component, and its bone marrow expression in acute myeloid leukemia (AML) has been suggested to have a prognostic impact for patients receiving intensive treatment.[1–3] These studies have mainly examined osteopontin mRNA expression of bone marrow cells [2,3] or protein levels in marrow plasma.[1,2] Osteopontin is thus involved in communication between cells. No previous studies have quantified the constitutive release of osteopontin by primary human AML cells, the variation of this release among patients and its possible prognostic impact. Primary AML cells derived from 79 patients (median age 67 years; range 18–87 years; 34 females and 45 males) were cultured in vitro either (i) alone in serum-free Stem Span medium (Stem Cells Technology, Vancouver, Canada) for 48 h;[4] or (ii) in transwell co-cultures (0.4 lm pore size) with normal bone marrow mesenchymal stem cells (MSCs) for 72 h before supernatant levels were measured by ELISA (R&D Systems, Abingdon, UK).[5] Our cohort represents a group of unselected patients with high numbers of circulating leukemia blasts in peripheral blood (> 15 10/L). As discussed previously, highly enriched AML cell populations can then be prepared by density gradient separation alone,[4,6] and the cells were stored in liquid nitrogen until use. Osteopontin mRNA levels were determined from global gene expression analyses.[7] The protein levels were quantified by applying the label-free proteomic methodology as described in detail elsewhere.[8] The constitutive osteopontin protein release for AML cells cultured alone varied, and detectable levels (>10 pg/mL) were seen only for 28 of the 79 patients (Figure 1(A)). Detectable constitutive release showed significant associations (Mann–Whitney U-test) with age 40 years (p1⁄4 .018), monocytic differentiation (FAB-M4/M5; p1⁄4 .002), and favorable cytogenetics (p< .001). The significances remained also when the two patients with acute promyelocytic leukemia (APL) were excluded. No significant associations with sample storage time or cell viability after 48 h of culturing were detected. We compared the overall survival for patients with and without detectable constitutive osteopontin release. The survival analysis included only those 31 patients (median age 55 years, range 18–75; 13 females and 18 males) that completed intensive induction chemotherapy (7 d of cytarabine infusion combined with 3 d anthracycline treatment) followed by intensive consolidation treatment (either high-dose cytarabine alone in single doses of 3 g/m or combination therapy based on cytarabine single doses of 1 g/m) and potentially allogeneic stem cell transplantation. The long-term overall survival was significantly increased for patients with detectable constitutive osteopontin release (i.e. supernatant levels >10 pg/mL) compared with patients showing undetectable levels (Figures 1(A/B)), p1⁄4 .002). An additional analysis where patients were classified into three groups – undetectable (osteopontin level <10 pg/mL, 16 patients), intermediate (10 pg/mL–1 ng/mL, 6 patients), and high release (> 1 ng/mL, 9 patients) – also reached statistical significance (p1⁄4 .003). Univariate analysis revealed Flt3-ITD (HR1⁄4 3.926; p1⁄4 .008) and undetectable osteopontin levels (HR1⁄4 4.407; p1⁄4 .005) as risk factors for reduced survival. However, in a multivariate Cox-regression analysis including the parameters age, FAB, Flt3-ITD, NPM1-mutations, CD34 expression, disease etiology, and osteopontin release – favorable cytogenetics were excluded due to few patients (only five non-APL patients received/completed intensive treatment, and two of them were lost from follow-up), undetectable osteopontin expression emerged as an independent factor for reduced survival (HR1⁄4 4.537; p1⁄4 .024). We then compared the osteopontin levels for AML cells cultured alone to those cells co-cultured with bone marrow MSCs from healthy donors. The constitutive release by AML cells alone (cultured in co-culture wells) showed significant correlation with the corresponding