Dataset Information

Refinement of paramagnetic bead–based digestion protocol for automatic sample preparation using an artificial neural network

ABSTRACT: Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead–based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (µg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile (ACN), and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 µg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 µg of beads for sample preparation and long-term digestion (16h) with 0.15 µg Lys-C and 2.5 µg trypsin (≈ 1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, rat bile, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. Notwithstanding, we must highlight the performance of this method in the preparation of a challenging biological fluid as bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction of digestion of bile proteins, our method allowed to identify exclusively 9.91% (x proteins), thus contributing to improving the coverage of the bile proteome.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Rattus Norvegicus (rat) Homo Sapiens (human) Escherichia Coli Arabidopsis Thaliana (mouse-ear Cress) Mus Musculus (mouse)

SUBMITTER: Sergio Ciordia

LAB HEAD: Fernando Corrales Izquierdo

PROVIDER: PXD048358 | Pride | 2024-05-31

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20230825_10_ANN_Manual_p2.raw	Raw
	20230825_13_ANN_OT2_p3.raw	Raw
	20230825_16_ANN_Manual_p3.raw	Raw
	20230825_19_ANN_OT2_p4.raw	Raw
	20230825_1_ANN_OT2_p1.raw	Raw

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Refinement of paramagnetic bead-based digestion protocol for automatic sample preparation using an artificial neural network.

Ciordia Sergio S Santos Fátima Milhano FM Dias João M L JML Lamas José Ramón JR Paradela Alberto A Alvarez-Sola Gloria G Ávila Matías A MA Corrales Fernando F

Talanta 20240323

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead-based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic samp ...[more]

PMID: 38569368

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Project description:Bile has gained increasing attention for its potential to reflect the overall health of the biliary system. Proteomic analysis of bile could deepen our understanding of the molecular pathophysiology of biliary disease and injury and provides a potential source of diagnostic biomarkers. With the emergence of normothermic machine perfusion (NMP) prior to liver transplantation, bile samples can be easily collected and analyzed. However, the unique composition of bile, including high concentrations of endogenous detergents and salts, can make application of proteomics challenging. In this study, we systematically evaluated a variety of available trypsin-digestion methods to optimize proteomics analysis of bile obtained from human NMP livers. Bile was collected from 12 human donor livers that were accepted for transplantation after NMP viability assessment. We performed tryptic digestion using 6 different methods: in-gel, in-solution, S-Trap, SMART, EasyPep, and filter-aided sample purification, with or without an additional precipitation step prior to digestion. Untargeted, data-independent acquisition proteomics was performed using an Exploris 480 mass spectrometer coupled with a FAIMS device. The methods were judged for total protein IDs, variation, and specific protein characteristics to determine the most optimal method. A total of 4650 unique proteins were identified across all samples and all methods. Methods involving precipitation identified substantially more proteins (4500 vs 3815, respectively). In-gel crude was the exception, identifying a comparable number of proteins to precipitated in-gel digest. We found minimal differences in the mass, cellular component, or hydrophobicity of identified proteins between methods. Intra-method variability was notably diverse, with in-gel, in-solution, and EasyPep outperforming other methods with considerably less variation. Age-related biological comparisons revealed significant disparities in protein abundances between methods, but younger donors consistently showed upregulation of metabolic-related processes compared to older donors. Older donors showed upregulation of immune response and cell cycle-related processes. The digestion method used for proteomic analysis of bile can heavily influence the results obtained. A method of protein purification appears to be essential for sufficient quality mass spectrometry runs and subsequent data. Sample processing speed, cost, cleanliness, and reproducibility should be carefully considered when selecting a protein digestion method for this difficult sample type.