Characterisation of the Tumour Proteome in Primary Extramedullary Multiple Myeloma Identifies Key Proteins Associated with Transendothelial Migration

Introduction: Extramedullary multiple myeloma (EMM) refers to the spread of clonal plasma cells to tissues extending outside of the bone marrow microenvironment. EMM is present at the time of diagnosis in 6-10% of patients, however, this increases to 13-26% in patients with disease progression and relapse. Cancer cells are suspected to spread to new tissues and organs via the circulatory system as a result of molecular changes that allow malignant cells to escape the bone marrow (BM). For example, downregulation of CXCR4 (C-X-C Motif Chemokine Receptor 4), an important factor in cellular homing to the BM, has frequently been reported to be linked to the EMM phenotype. In addition, the majority of patients presenting with EMM have highly complex cytogenetic abnormalities and high-risk cytogenetic markers such as t(14;16). As EMM is an indicator of a more aggressive disease, more intensive treatment, including combination chemotherapy, is often recommended. Many of the underlying molecular mechanisms accompanying EMM are yet to be characterised. Our mass-spectrometry (MS)-based proteomic study provides insight into the unique molecular mechanisms associated with EMM, identifying key proteins linked to the progression of medullary multiple myeloma (MM) to EMM. Methods: Label-free liquid chromatography mass spectrometric analysis of age and gender matched medullary MM (n=8) and EMM (n=9) bone-marrow derived mononuclear cells (MNCs) was carried out using a Thermo Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Proteome Discoverer 2.2 using Sequest HT (Thermo Fisher Scientific) and a percolator were employed for the identification of peptides and proteins. For protein identification, the following search parameters were used: (i) 0.02 Da for MS/MS mass tolerance, (ii) 10 ppm for peptide mass tolerance, (iii) variable modification settings for methionine oxidation, (iv) fixed modification settings in relation to carbamido-methylation and (v) tolerance for up to two missed cleavages. Peptide probability was set to high confidence. Datasets were imported into Progenesis QI (version 2.0) software for further analysis. Data was filtered based on an ANOVA p-value of ≤0.05, fold change >1.5 between experimental groups, and proteins with ≥1 unique peptides contributing to the identification. Proteins with less than 70% valid values were removed from the analysis. G:profiler and STRING were utilized for functional enrichment and the characterisation of protein interaction patterns. Results: Our quantitative MS-based proteomic analysis identified a total of 492 proteins with significantly altered abundances between EMM and MM bone marrow MNC. Of these significant proteins, 275 were found to be increased in EMM compared to medullary MM and 217 were found to be decreased in EMM compared to medullary MM. Hierarchical clustering was performed to highlight the proteomic profile associated with extramedullary disease (Figure 1A). KEGG pathway analysis and gene ontology (GO) analysis of proteins found to be increased in EMM indicated an increase in proteins associated with cell adhesion, invasion, and migration pathways (Figure 1B). Interestingly, several proteins involved in leukocyte transendothelial migration were significantly increased in EMM indicating their potential involvement in the dissemination of MM cells from the bone marrow microenvironment to distal tissues (Figure 1C). Among the proteins found to be involved in this biological pathway was junctional adhesion molecule-A (F11R), a protein previously reported to play a role in EMD pathophysiology [1]. Other proteins involved in MM invasion and migration including Rho-associated protein kinase 2 (ROCK2), Ras-related C3 botulinum toxin substrate 1 (Rac1) and platelet endothelial cell adhesion molecule (PECAM-1) were significantly increased in EMM. Conclusion: Using high-resolution mass spectrometry to characterise the tumour proteome of MM patients with extramedullary disease, we have identified a significant increase in the abundance of proteins associated with leukocyte transendothelial invasion in primary EMM samples. Our study provides further insight into the molecular mechanisms within EMM and thus holds potential to enhance current efforts to provide a more personalised therapeutic approach for EMM patients. References: [1] A.G. Solimando et al., Blood 2018; 132 (Supplement 1): 4455. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Naive Bayes classification model for isotopologue detection in LC-HRMS data

Isotopologue identification or removal is a necessary step to reduce the number of features that need to be identified in samples analyzed with non-targeted analysis. Currently available approaches rely on either predicted isotopic patterns or an arbitrary mass tolerance, requiring information on the molecular formula or instrumental error, respectively. Therefore, a Naive Bayes isotopologue classification model was developed that does not depend on any thresholds or molecular formula information. This classification model uses elemental mass defects of six elemental ratios and can successfully identify isotopologues in both theoretical isotopic patterns and wastewater influent samples, outperforming one of the most commonly used approaches (i.e., 1.0033 Da mass difference method - CAMERA).

Justification scheme for milling waffle background

In the manufacture of dry and fuel compartments of missiles ensuring weight tolerance is a priority. In practice, this is ensured by the introduction of lightweight materials together with the optimal design of the supporting set of ribs (waffle background) of orthotropic shells and waffle shells. The manufacturing technology of which is difficult and costly in view of the difficulty of ensuring the accuracy and perfection of the shape of the waffle background cell. In modern conditions, when manufacturing a waffle shell for a fuel tank, reinforcement processing is carried out by milling on a monolithic blank using specialized machine systems. At the same time, the choice of the waffle background processing scheme remains an urgent issue: on a flat panel or on a bent shell. The problem of selection arises due to the requirements for the accuracy of the waffle background. It is important to maintain the part mass tolerance and geometric perfection of the cell shape. All this generally affects the perception of design loads and the carrying capacity of the rocket. At the same time, the methods and processes of processing the waffle background should be productive and economically feasible. The proposed justification will make it possible at the start of the design of new promising products to more accurately determine the technological and production costs necessary for the production of parts with a waffle background with given parameters and accuracy.

An Algorithm to Improve the Speed of Semi- and Non-Specific Enzyme Searches in Proteomics

Background: The identification of non-specifically cleaved peptides in proteomics and peptidomics poses a significant computational challenge. Current strategies for the identification of such peptides are typically time consuming and hinder routine data analysis. Objective: We aimed to design an algorithm that would improve the speed of semi- and non-specific enzyme searches and could be applicable to existing search programs. Method: We developed a novel search algorithm that leverages fragment-ion redundancy to simultaneously search multiple non-specifically cleaved peptides at once. Briefly, a theoretical peptide tandem mass spectrum is generated using only the fragment-ion series from a single terminus. This spectrum serves as a proxy for several shorter theoretical peptides sharing the same terminus. After database searching, amino acids are removed from the opposing terminus until the observed and theoretical precursor masses match within a given mass tolerance. Results: The algorithm was implemented in the search program MetaMorpheus and found to perform an order of magnitude faster than the traditional MetaMorpheus search and produce superior results. Conclusion: We report a speedy non-specific enzyme search algorithm which is open-source and enables search programs to utilize fragment-ion redundancy to achieve a notable increase in search speed.

Stand for measuring the volume of the blank using a laser sensor

The aim of the article is to present a method of measuring the volume of a blank using a laser sensor and a complete measuring stand. In the analyzed part of the manufacturing process of water fittings, the brass rod is cut on blanks of a given weight using CNC sawing machine. Due to the applied flashless forging technology, high cutting precision is required, understood as a relative mass tolerance of the blank of less than 0.1%. In this case, it becomes important to measure the geometry of the rod to calculate its volume – to correct the length of the blank. The natural way to calculate the volume of rotational solids with complex shapes is to determine the definite integral of the curve describing the outer surface of the solid. However, in the case of solids with geometrical errors this function may vary with the angle of rotation of the solid. In the case of real systems, in which the measurement results are a discrete sequence of numbers, it is natural to choose numerical integration. The paper presents the use of a laser sensor moved around the axis and along the rod to measure its volume and the concept of a complete measuring stand.