Optimization of the workflow for deep CSF proteome analysis by enrichment of extracellular vesicles

ABSTRACT: Proteome analysis of cerebrospinal fluid is associated with many of the same challenges that has to be faced when analyzing other biofluids like plasma or urine. Most importantly is the high dynamic range of protein concentrations, which makes it difficult to quantify more than a thousand proteins in a singleshot analysis of a tryptic digest of a raw sample. In addition, cerebrospinal fluid—especially when collected from children—is a much more scarce and precious sample as compare to urine and plasma. The aim of this work therefore was to find the optimal way to process and analyze cerebrospinal fluid from a minimal amount of sample and still obtain the deepest possible cerebrospinal fluid proteome. The work shows that enrichment of extracellular vesicles by ultracentrifugation increases the complexity of the cerebrospinal fluid proteome by two-fold or more. Thus, by following the enrichment protocol, more than two thousand proteins can routinely be quantified reproducibly by label-free quantification and data independent acquisition (DIA) in single-shot LC-MSMS runs of less than one hour. The project also present an optimized workflow for cerebrospinal fluid proteomics that enables a high-throughput method with a short chromatographic gradient (12 min) allowing analysis of hundred samples per day and still obtaining deep proteome coverage, especially when including a study-specific spectral library generated by repeated injection and gas-phase fractionation of pooled samples.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cerebrospinal Fluid

DISEASE(S): Acute Leukemia

SUBMITTER: Ole Østergaard  

LAB HEAD: Jesper Velgaard Olsen

PROVIDER: PXD052666 | Pride | 2024-10-16

REPOSITORIES: Pride