Dataset Information

Proteome-wide non-cleavable crosslink identification with MS Annika 3.0 reveals the structure of the C. elegans Box C/D complex

ABSTRACT: The field of crosslinking mass spectrometry has seen substantial advancements over the past decades, enabling the structural analysis of proteins and protein-complexes and serving as a powerful tool in protein-protein interaction studies. However, data analysis of large non-cleavable crosslink studies is still a mostly unsolved problem due to its n-squared complexity. We here introduce a novel algorithm for the identification of non-cleavable crosslinks implemented in our crosslinking search engine MS Annika that is based on sparse matrix multiplication and allows for proteome-wide searches on commodity hardware. Application of this new algorithm enabled us to employ a proteome-wide search of C. elegans nuclei samples and we were able to uncover previously unknown protein interactions and conclude a comprehensive structural analysis that provides a detailed view of the Box C/D complex, enhancing our understanding of its assembly and functional dynamics. Our findings provide valuable insights into the intricate regulation of cellular homeostasis and immune responses, which are conserved across species, including humans. Moreover, our algorithm will enable researchers to conduct similar studies that were previously unfeasible.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Caenorhabditis Elegans

TISSUE(S): Gonad, Nucleate Cell

SUBMITTER: Micha Johannes Birklbauer

LAB HEAD: Viktoria Dorfer

PROVIDER: PXD055488 | Pride | 2024-12-19

REPOSITORIES: Pride

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			Action	DRS
	20230612_E3_RSLC6_Mueller_MS_TechHub_IMP_THIDVJSSG004_Celegans_Nulcei_S1_DSG_SEC105_1ug_FAIMS_001.raw	Raw
	20230612_E3_RSLC6_Mueller_MS_TechHub_IMP_THIDVJSSG004_Celegans_Nulcei_S1_DSG_SEC10_1ug_FAIMS_001.raw	Raw
	20230612_E3_RSLC6_Mueller_MS_TechHub_IMP_THIDVJSSG004_Celegans_Nulcei_S1_DSG_SEC115_500ng_FAIMS_001.raw	Raw
	20230612_E3_RSLC6_Mueller_MS_TechHub_IMP_THIDVJSSG004_Celegans_Nulcei_S1_DSG_SEC11_500ng_FAIMS_001.raw	Raw
	20230612_E3_RSLC6_Mueller_MS_TechHub_IMP_THIDVJSSG004_Celegans_Nulcei_S1_DSG_SEC12_500ng_FAIMS_001.raw	Raw

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Project description:We investigated gene expression levels in Heliconius erato butterflies with divergent wing patterns across a 656KB genomic interval linked to the red color pattern wing polymorphism. This included comparison of expression between two H. erato color pattern populations (H. e. petiverana and a H.e. etylus x H. himera hybrid) across three sections of the forewing that differed in pigmentation (the basal, mid, and distal wing sections) and five different stages of pupal development (Day 1, 3, 5 pupae and ommochrome and melanin pigmentation stages). These results allowed us to determine whether certain genes in this interval were differentially expressed between the wing pattern elements, and, therefore, potentially responsible for adaptive color pattern variation in these butterflies. Forewings from a total of 29 individuals, covering three biological replicates of five developmental time points for each of the two H. erato distinct phenotypes were dissected, with the only exception being that there were only two replicates of the day 1 hybrid phenotype. Individuals were reared at 25˚C and dissected at the following stages: a) day 1 = 12 hr after pupation; b) day 3 = 60 hr after pupation; c) day 5 = 108 hr after pupation; d) early ommochrome = ~ 156 hr after pupation when red scales in forewing partially mature, showing a pale orange color; and e) early melanin = ~ 180 hr after pupation melanic scales begin to turn black and are present primarily at the center of the wing. Using wing veins as landmarks, each forewing was cut into three sections corresponding to the color pattern boundaries: basal (F1), middle (F2), and apical (F3). A eight custom-designed Roche NimbleGen 12x135K format microarrays with probes spanning a 656,307bp genomic region (Roche NimbleGen Inc., Madison, Wisconsin, United States) were used to hybridize double stranded cDNA from 87 tissue samples. Repetitive sequence elements found more than five times across all currently available H. erato genomic sequences, including the probed region as well as additional genomic BAC sequences, were masked from the tiling region. The remaining unmasked non-repetitive genomic sequences were tiled using 60 bp probes staggered every 13 bp on average, with slight modifications to ensure probe quality, for a total of 48,547 probes. Microarry design and printing was performed by Roche NimbleGen. cDNA labeling, hybridization, and array scanning was performed by the City of Hope Microarray Facility (Duarte, California, United States). In addition to the probes from the red color pattern intervals, the arrays also include 40,763 probes across two other genomic intervals not addressed in this study, 45,046 probes representing 12450 transcripts from a recent transcriptome assembly at 1-6X coverage, and 3248 random probes. Results from the transcriptome and other color pattern intervals will be published separately, however, we analyzed all probes together for array normalization and quality control.

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Proteome-wide non-cleavable crosslink identification with MS Annika 3.0 reveals the structure of the C. elegans Box C/D complex

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