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Centrifugal microfluidic automation of the protein aggregation capture workflow for robust mass spectrometry-based proteomics

ABSTRACT: Proteomic sample preparation for liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly addressed by automated approaches. However, in clinical settings, where only a limited amount of samples has to be processed in a standardized and reproducible manner, no fully automized workflow was provided yet. remains a major bottleneck for the broader adoption of clinical and translational proteomics due to its labor-intensive and error-prone nature. Here, we present the AutoPAC-Disk, a centrifugal microfluidic implementation of a protein aggregation capture (PAC) sample preparation workflow for bottom-up proteomics. The AutoPAC-Disk features a bead-packed column with a miniaturized digestion volume of 14 µL and pre-stored organic solvents. A comparative evaluation using HEK293 cell lysates revealed higher identification rates than both a manual commonly used reference workflow and a its semi-automated robotic system implementation (KingFisher), yielding 50 % and 37 % more peptide identifications and 23 % and 10 % more protein identifications, respectively. These gains in peptide identifications translated into increased sequence coverage of the identified proteins. Quantitative reproducibility was high for all approaches, with medianOur AutoPAC-Disk resulted in a similar exhibits high quantitative reproducibility with protein group intensity coefficients of variation below 10 %, similar to the other well -established workflows. Further analysis revealed that the AutoPAC-Disk predominantly enabled additional protein identifications in the low-abundance range. Together, these results provide a proof of concept that microfluidic automation, including the successful on-disk pre-storage of reagents and buffers, can substantially simplify proteomic sample preparation by enabling a high degree of automation and minimal user interaction. This approach lowers the operational barrier for personnel with limited experience in proteomic sample preparation and represents a promising strategy for future applications in clinical and translational proteomics within the field of precision medicine.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Johanna Wallner

LAB HEAD: Hannes Hahne

PROVIDER: PXD072920 | Pride | 2026-06-29

REPOSITORIES: Pride

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			Action	DRS
	C_0388_M_P11807_S124403_A01_MnDIA-LFQ_F00_R01.raw	Raw
	C_0388_M_P11807_S124404_B01_MnDIA-LFQ_F00_R01.raw	Raw
	C_0388_M_P11807_S124405_C01_MnDIA-LFQ_F00_R01.raw	Raw
	C_0388_M_P11807_S124406_A02_MnDIA-LFQ_F00_R01.raw	Raw
	C_0388_M_P11807_S124407_B02_MnDIA-LFQ_F00_R01.raw	Raw

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Project description:Cells rely on the Unfolded Protein Response (UPR) to maintain ER homeostasis (proteostasis) when faced with elevated levels of misfolded and aggregated proteins, which can hinder the secretion of properly folded proteins. The UPR is composed of three main branches—ATF6, IRE1, and PERK—that coordinate the synthesis of proteins involved in folding, trafficking, and degradation of nascent proteins to restore ER function. Dysregulation of the UPR is linked to numerous diseases, including neurodegenerative disorders, cancer, and diabetes. Despite its importance, identifying UPR targets has been challenging due to their heterogeneous induction, which varies by cell type and tissue. Additionally, defining the magnitude and range of UPR-regulated genes is difficult because of intricate temporal regulation, feedback between UPR branches, and extensive cross-talk with other stress-signaling pathways. To comprehensively identify UPR-regulated proteins and determine their branch specificity, we developed a data-independent acquisition (DIA) liquid-chromatography mass spectrometry (LC-MS) pipeline. Our optimized workflow improved protein identifications of low-abundant UPR proteins and leveraged an automated SP3-based protocol on the Biomek i5 liquid handler for label-free peptide preparation. Using engineered stable cell lines that enable selective pharmacological activation of each UPR branch without triggering global UPR activation, we identified branch-specific UPR proteomic targets. These targets were subsequently applied to investigate proteomic changes in multiple BRAF-mutant melanoma cell lines, representing different genetic backgrounds, treated with a BRAF inhibitor (PLX4720, i.e., vemurafenib). Our findings revealed differential regulation of the XBP1s branch of the UPR in the BRAF-mutant melanoma cell lines after PLX4720 treatment, likely due to calcium activation, suggesting that the UPR plays a role as a non-genetic mechanism of drug tolerance in melanoma. In conclusion, the validated branch-specific UPR proteomic targets identified in this study provide a robust framework for investigating this pathway across different cell types, drug treatments, and disease conditions in a high-throughput manner.

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Centrifugal microfluidic automation of the protein aggregation capture workflow for robust mass spectrometry-based proteomics

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