Proteomic Spanish User Group Meeting 2024

15:00 – Dr. Quentin Enjalbert – PreOmics

Welcome & Introduction

15:10 – Sònia Jarió Ruana - Mass Spectrometry & Proteomics Core Facility at IRB Barcelona

Analysis of multiple mouse tissue proteomes using PreOmics’s BeatBox-iST methodology

15:30 – Eduard Sabido/Eva Borras - CGR Barcelona

Implementation of the ENRICH family kits for biofluids

15:50 – Manuel Fuentes - Proteomics Group and Pathology Team

PreOmic´s BeatBox-iST approaches where Pathology meets Proteomics

16:10 – Ana Montero Calle - Functional Proteomics Unit, Chronic Disease Programme (UFIEC), Instituto de Salud Carlos III, Madrid

Advancing Plasma Proteomics: Leveraging DIA and PRM Approaches on Depleted Samples with the Orbitrap Astral for Biomarker Detection

16:30 – Teresa García-Berrocoso - R&D department at CORNEA PROJECT S.L. - Sitges (Barcelona)

Methods comparison for protein extraction, solubilization and digestion from paucicellular human clinical specimens.

16:50 – Question and answer session

Open questions to the speakers and PreOmics team (in Spanish/English language)

The present study is a multidisciplinary research that includes the compilation of the first E3 ubiquitin ligases Atlas of aging. The expression of the ~600 ubiquitin E3 ligases (E3s) was interrogated by quantitative proteomics in a panel of tissues from young (6 months old) versus naturally aged (24 months old) mice. In order to perform global and quantitative proteomics tissue analyses, the BeatBox-iST protocol (iST Sample Preparation Kit 96x, PreOmics) was optimized to achieve compatibility with our tissue-dependent downstream protein digestion workflow. NanoLC-MS/MS data was acquired on an Orbitrap Eclipse Tribrid mass spectrometer (ThermoFisher Scientific, San Jose, CA) connected to an EVOSEP One (EVOSEP, Odense, Denmark) by DIA strategies.

Biofluids are a valuable resource for studying diseases, aiding in biomarker identification and enhancing our understanding of disease mechanisms; however, their proteome profiling via mass spectrometry encounters challenges due to the broad dynamic range of protein abundances. Recent advancements in mass spectrometry, along with commercial solutions like the ENRICH-iST kit by PreOmics, which is designed to improve the detection of low-abundance proteins, have enhanced the processing of liquid biopsies. In this study, we adapted the ENRICH-iST protocol for cerebrospinal fluid (CSF), evaluating its sensitivity and reproducibility across various sample amounts and mass spectrometry platforms. The results reveal a substantial increase in proteome coverage and depth while maintaining technical reproducibility, thereby facilitating precise profiling of CSF samples for neurological disease research.

As a joint project between the Proteomics Unit at Biomedical Research Center Salamanca (IBSAL) and the Molecular Pathology Lab and coordination node of the regional Biobank of Oncological tissues ( Biobanco en Red de Enfermedades Oncológicas de Castilla y León).

In order to perform quantitative proteomics tissue analysis; an optimal experimental workflow has been designed and developed which is based on BeatBox -iST ( iST sample preparation kit, PreOmics). Different solid tumors ( breast, lung, ….) in several formats ( FPPE, LCM,….) have been analyzed by these workflows. NanoLC-MS/MS data has been obtained by TIMS-TOF Pro-2 mass spectrometer (Bruker,Germany) connected to an EVO-SEP One ( EVOSEP, Odense, Denmark) by DIA-PASEF.

Non-infectious chronic diseases are currently the most common cause of disability worldwide, and responsible for 74% of total deaths worldwide. Most chronic diseases are underdiagnosed or diagnosed at late stages, primarily due to the late onset of their first clinical symptoms. Thus, the identification of biomarkers in minimally invasive biological fluids with diagnostic ability of these pathologies is mandatory to improve their diagnosis, treatment, and progression.

The proteomics analyses of plasma or serum samples plays a crucial role for the identification of biomarkers of chronic diseases that could be implemented in clinical routine to improve their diagnosis, treatment, and progression. However, mass spectrometry analyses yet face challenges due to the wide dynamic range in protein concentration of these samples. Thus, the depletion of high-abundant proteins, such as albumin or immunoglobulins, is essential to reduce sample complexity and enhance the detection of low-abundant proteins.

We here aimed at identifying plasma biomarkers for colorectal cancer (CCR) and Alzheimer’ disease (AD), two of the most common chronic diseases worldwide. To that end, plasma samples were depleted using three different strategies to enrich them in low-abundant proteins that might be useful as biomarkers of these diseases: immunoaffinity chromatography, ENRICH-iST kit (PREOMICS), and Mag-Net (Resyn Biosciencea) methods. Them, depleted and 1 µL of neat plasma samples were trypsin digested and analyzed using data independent acquisition (DIA) in the Orbitrap Astral and Orbitrap Eclipse Tribrid mass spectrometers, with 15-minutes and 2-hours gradients, respectively. Nearly 4000 plasma proteins were identified in the plasma samples depleted by the non-conventional immunoaffinity methods with the Astral mass spectrometer. Additionally, the efficacy of plasma enrichment methods was assessed using parallel reaction monitoring (PRM) in the Orbitrap Astral and Orbitrap Eclipse Tribrid mass spectrometers, with 15-minutes and 2-hours gradients, respectively, to detect previously described CRC and AD plasma biomarkers. The superior performance of the combination of plasma depletion and the Orbitrap Astral mass spectrometer was further demonstrated for targeted proteomics analyses.

The optimization of sample preparation techniques, particularly those focused on enriching low-abundance proteins in plasma, is crucial for enhancing the depth and sensitivity of mass spectrometry-based proteomic analyses. This approach enables the detection of a wider array of proteins and potential biomarkers associated with various diseases. Such strategies benefit both discovery-driven and targeted proteomic studies, offering critical insights into disease mechanisms and identifying promising diagnostic markers.

Biological samples containing scarce starting material are frequently common in clinical settings and pose a challenge in untargeted human LC-MS-based proteomics studies.

In a collaborative precision medicine project between CORNEA PROJECT and IMIBIC R&D teams, 8 different methodologies for sample preparation were assessed, combining different cell lysis buffers, mechanical cell disruption methods and protein digestion approaches. Biological triplicates from real clinical specimens and negative controls were used in each combination tested and equal peptide amount was analyzed by DIA-PASEF technology on a timsTOF Flex (Bruker) coupled to an EvosepOne (Evosep).

Performance of each method combination was evaluated based on purified peptide quantification, the number of peptide and protein identifications and proteome coverage. Moreover, reproducibility, easiness of use, processing time and throughput capabilities were also considered. After this feasibility study, the best protocol combination was selected for upcoming clinical studies.

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Proteomics Spanish User Group Meeting

OVERVIEW

Proteomics Spanish User Group Meeting

AGENDA

OCTOBER 1, 2024

SPEAKERS & ABSTRACTS

Sònia Jarió Ruana - Mass Spectrometry & Proteomics Core Facility at IRB Barcelona

Eduard Sabido - CGR Barcelona

Manuel Fuentes - Proteomics Facility at Institute of BioMedical Research Salamanca (IBSAL) M Carmen García Macias - BioBanco en Red de Enfermedades Oncológicas (BEOCyL)

Ana Montero Calle - Functional Proteomics Unit, Chronic Disease Programme (UFIEC), Instituto de Salud Carlos III, Madrid

Teresa García-Berrocoso - R&D department at CORNEA PROJECT S.L. - Sitges (Barcelona) Ángela Peralbo-Molina - Mass Spectrometry unit at IMIBIC - Córdoba

Dr. Quentin Enjalbert - PreOmics