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Systematic evaluation of experimental parameters for sensitive phosphoproteomics analysis

ABSTRACT: The field of phosphoproteomics has been revolutionized in the last years, with enhanced efficiency and sensitivity, now enabling deep phosphoproteomic analysis single-shot from minimal peptide inputs. In this study, we rigorously evaluate the effects of various experimental parameters in automated Zirconium-IMAC (Zr-IMAC) based phosphoproteomic sample preparation with focus on low peptide input samples. Assessing metrics such as number of identified phosphopeptides, selectivity, phosphosite localization scores and relative purification of multiply phosphorylated phosphopeptides, we were able to pinpoint key influencing variables. Determination of the optimal glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide to beads ratio, binding time, sample volume and loading buffer volume, allowed us to confidently localize up to ~15,800 phosphopeptides using 30 µg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost the depth of the analysis, and most importantly, whether pooling fractions into a single-LCMS analysis also increases the phosphoproteome depth. As a result, we present a novel strategy based on incremental addition of beads and subsequent fraction pooling, which can boost by 17% the phosphoproteome coverage when compared to standard enrichment.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ana Martinez-Val

LAB HEAD: Jesper V. Olsen

PROVIDER: PXD045601 | Pride | 2024-03-26

REPOSITORIES: Pride

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