Identification of LMAN1- and SURF4-Dependent Secretory Cargoes.

Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles and tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells, whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif..

Solid-Phase Compatible Silane-Based Cleavable Linker Enables Custom Isobaric Quantitative Chemoproteomics.

Mass spectrometry-based chemoproteomics has emerged as an enabling technology for functional biology and drug discovery. To address limitations of established chemoproteomics workflows, including cumbersome reagent synthesis and low throughput sample preparation, here, we established the silane-based cleavable isotopically labeled proteomics (sCIP) method. The sCIP method is enabled by a high yielding and scalable route to dialkoxydiphenylsilane fluorenylmethyloxycarbonyl (DADPS-Fmoc)-protected amino acid building blocks, which enable the facile synthesis of customizable, isotopically labeled, and chemically cleavable biotin capture reagents. sCIP is compatible with both MS1- and MS2-based quantitation, and the sCIP-MS2 method is distinguished by its click-assembled isobaric tags in which the reporter group is encoded in the sCIP capture reagent and balancer in the pan cysteine-reactive probe. The sCIP-MS2 workflow streamlines sample preparation with early stage isobaric labeling and sample pooling, allowing for high coverage and increased sample throughput via customized low cost six-plex sample multiplexing. When paired with a custom FragPipe data analysis workflow and applied to cysteine-reactive fragment screens, sCIP proteomics revealed established and unprecedented cysteine-ligand pairs, including the discovery that mitochondrial uncoupling agent FCCP acts as a covalent-reversible cysteine-reactive electrophile.

Statistical Detection of Differentially Abundant Proteins in Experiments with Repeated Measures Designs and Isobaric Labeling.

Repeated measures experimental designs, which quantify proteins in biological subjects repeatedly over multiple experimental conditions or times, are commonly used in mass spectrometry-based proteomics. Such designs distinguish the biological variation within and between the subjects and increase the statistical power of detecting within-subject changes in protein abundance. Meanwhile, proteomics experiments increasingly incorporate tandem mass tag (TMT) labeling, a multiplexing strategy that gains both relative protein quantification accuracy and sample throughput. However, combining repeated measures and TMT multiplexing in a large-scale investigation presents statistical challenges due to unique interplays of between-mixture, within-mixture, between-subject, and within-subject variation. This manuscript proposes a family of linear mixed-effects models for differential analysis of proteomics experiments with repeated measures and TMT multiplexing. These models decompose the variation in the data into the contributions from its sources as appropriate for the specifics of each experiment, enable statistical inference of differential protein abundance, and recognize a difference in the uncertainty of between-subject versus within-subject comparisons. The proposed family of models is implemented in the R/Bioconductor package MSstatsTMT v2.2.0. Evaluations of four simulated datasets and four investigations answering diverse biological questions demonstrated the value of this approach as compared to the existing general-purpose approaches and implementations.