All-Ion Monitoring-Directed Low-Abundance Protein Quantification Reveals CALB2 as a Key Promoter in Hepatocellular Carcinoma Metastasis

Because of the wide abundance range
of the proteome, achieving
high-coverage quantification of low-abundance proteins is always a
major challenge. In this study, a complete pipeline focused on all-ion
monitoring (AIM) is first constructed with the concept of untargeted
parallel-reaction monitoring, including the seamless connection of
protein sample preparation, liquid chromatography mass spectrometry
(LC-MS) acquisition, and algorithm development to enable the in-depth
quantitative analysis of low-abundance proteins. This pipeline significantly
improves the reproducibility and sensitivity of sample preparation
and LC-MS acquisition for low-abundance proteins, enabling all the
precursors ions fragmented and collected. Contributed by the advantages
of the AIM method with all the target precursor acquisition by the
data-dependent acquisition (DDA) approach, together with the ability
of data-independent acquisition to fragment all precursor ions, the
quantitative accuracy and precision of low-abundance proteins are
greatly enhanced. As a proof of concept, this pipeline is employed
to discover the key differential proteins in the mechanism of hepatocellular
carcinoma (HCC) metastasis. On the basis of the superiority of AIM,
an extremely low-abundance protein, CALB2, is proposed to promote
HCC metastasis in vitro and in vivo. We also reveal that CALB2 activates the TRPV2-Ca2+-ERK1/2
signaling pathway to induce HCC cell metastasis. In summary, we provide
a universal AIM pipeline for the high-coverage quantification of low-abundance
functional proteins to seek novel insights into the mechanisms of
cancer metastasis.