Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes

Proteins often assemble
into multiprotein complexes for carrying
out their biological functions. Affinity purification combined with
mass spectrometry (AP-MS) is a method of choice for unbiasedly charting
protein complexes. Typically, genetically tagged bait protein and
associated proteins are immunoprecipitated from cell lysate and subjected
to in-gel or on-bead digestion for MS analysis. However, the sample
preparation procedures are often time-consuming and skipping reduction
and alkylation steps results in incomplete digestion. Here, by seamlessly
combining AP with the simple and integrated spintip-based proteomics
technology (SISPROT), we developed an integrated AP-MS workflow for
simultaneously processing more than 10 AP samples from cells cultured
in six-well plates in 2 h. Moreover, we developed a quantitation-based
data analysis workflow for differentiating potential interacting proteins
from nonspecific interferences. The AP-SISPROT ensures high digestion
efficiency especially for large transmembrane proteins such as EGFR
and high quantification precision for profiling temporal interaction
network of key EGFR signaling protein GRB2 across four time points
of EGF treatment. More importantly, the integration feature allows
minimum sample lose and helps the development of an ideal AP-MS workflow
for studying endogenous protein complexes by the CRISPR Cas9 technology
for the first time. By generating endogenously expressed bait protein
fused with affinity tag, protein complexes associated with endogenous
Integrin-linked kinase (ILK) was identified with much higher selectivity
as compared with overexpressed and tagged ILK. The AP-SISPROT technology
and its combination with CRISPR Cas9 technology should be generally
applicable for studying protein complexes in a more efficient and
physiologically relevant manner.