A mouse SWATH-MS reference spectral library enables deconvolution of species-specific proteomic alterations in human tumour xenografts

Abstract

SWATH-mass spectrometry (MS) enables accurate and reproducible proteomic profiling in multiple model organisms including the mouse. Here, we present a comprehensive mouse reference spectral library (MouseRefSWATH) that permits quantification of up to 10,597 proteins (62.2% of the mouse proteome) by SWATH-MS. We exploit MouseRefSWATH to develop an analytical pipeline for species-specific deconvolution of proteomic alterations in human tumour xenografts (XenoSWATH). This method overcomes the challenge of high sequence similarity between mouse and human proteins, facilitating the study of host microenvironment-tumour interactions from ‘bulk tumour’ measurements. We apply the XenoSWATH pipeline to characterize an intraductal xenograft model of breast ductal carcinoma in situ and uncover complex regulation consistent with stromal reprogramming, where the modulation of cell migration pathways is not restricted to tumour cells but also operates in the mouse stroma upon progression to invasive disease. MouseRefSWATH and XenoSWATH open new opportunities for in-depth and reproducible proteomic assessment to address wide-ranging biological questions involving this important model organism.

Data-Independent Acquisition Enables Robust Quantification of 400 Proteins in Non-Depleted Canine Plasma

“…These parameters add confidence to the protein and peptide quantifications among different samples and within replicates of the samples in the experiment. In this study, a spectral library was generated using information (in regards to instrumentation, and software analysis) from previously published DDA libraries in humans [ 14 ] and model animals [ 19 , 20 , 72 ]. Detailed information on generating spectral libraries, data acquisition, and DIA analysis has been discussed elsewhere [ 73 , 74 ].…”

“…In this acquisition mode, all peptides entering a mass spectrometer (irrespective of their abundance) will be fragmented (in consecutive isolation windows ranging between 10–50 da) within a given mass window (400–1200 m / z ) [ 17 ], which enables reproducible quantitation of peptides from complex sample mixtures. However, SWATH data analysis relies on the availability of a highly curated DDA based reference spectral library, which should be well-annotated with a genome and contains information about peptide sequences, their chromatographic elution times, and characteristic product ions [ 18 ], and a significant community effort has been made to develop relevant resources for several species [ 19 , 20 ]. With the availability of spectral libraries in humans [ 21 ], SWATH is now extensively used to explore the pathophysiology of diseases in humans [ 22 , 23 ].…”

Dietary-challenged mice with Alzheimer-like pathology show increased energy expenditure and reduced adipocyte hypertrophy and steatosis

“…Raw data were processed using DIA-NN 1.7.12 [ 56 ] , scan window size set to 14 and MS2 and MS1 mass accuracies to 20 and 10 ppm, respectively. A project-independent public spectral library [ 57 ] and mouse UniProt (UP000000589) were used for annotation. The library was automatically refined based on the dataset, global q=0.01 (using the “Generate spectral library” option in DIA-NN) [ 58 ].…”

Communication, construction, and fluid control: lymphoid organ fibroblastic reticular cell and conduit networks

“…The ECM is present throughout all developing and adult tissues, providing not only structure and scaffolding but also biochemical signals and mechanical cues to direct the behavior of residing cells [34]. There is broad heterogeneity in matrix composition and arrangement between tissues [35]. The ECM structures within SLOs are atypical, however, compared to other organs where fibroblasts reside surrounded by matrix structures; in SLOs the secreting fibroblasts unilaterally deposit and bundle matrix basolaterally into the conduit network they ensheath [28].…”