Combining Rapid Data Independent Acquisition and CRISPR Gene Deletion for Studying Potential Protein Functions: A Case of HMGN1

Abstract

CRISPR‐Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non‐histone chromosomal protein HMG‐14 (HMGN1) in regulating chromatin structure and tumor immunity, gene knockout of HMGN1 is performed by CRISPR in cancer cells and the following proteomic regulation events are studied. In particular, DIA mass spectrometry (DIA‐MS) is utilized, and more than 6200 proteins (protein‐ FDR 1%) and more than 82 000 peptide precursors are reproducibly measured in the single MS shots of 2 h. HMGN1 protein deletion is confidently verified by DIA‐MS in all of the clone‐ and dish‐ replicates following CRISPR. Statistical analysis reveals 147 proteins change their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. It is suggested that DIA‐MS can be reliably used as a rapid, robust, and cost‐effective proteomic‐screening tool to assess the outcome of the CRISPR experiments.

Isoform‐resolved correlation analysis between mRNA abundance regulation and protein level degradation

“…Further, increasing MS analytical sensitivity is essential for identifying new AS‐specific peptides (Schreiner et al , ). Here, we employed an optimized, sensitive DIA‐MS method on a high‐resolution Orbitrap platform (Bruderer et al , ; Amon et al , ; Mehnert et al , ) and re‐measured proteomic samples used in a previous multi‐omic study, in which heterogeneity of HeLa cells across different research laboratories was analyzed (Liu et al , ). These HeLa cell lines were shown to harbor a considerable heterogeneity on mRNA, protein, and protein degradation stemming from copy number variations (CNV) accumulated due to genomic instability and clonal effects (Liu et al , ).…”

“…These HeLa cell lines were shown to harbor a considerable heterogeneity on mRNA, protein, and protein degradation stemming from copy number variations (CNV) accumulated due to genomic instability and clonal effects (Liu et al , ). Herein, using the same MS sample sets of HeLa strains, the identical spectral library that contains mass spectrometric assays for 10,000 human proteins (Rosenberger et al , ), and the same statistical threshold [1% peptide and 1% protein FDR (Rosenberger et al , )], we were able to identify a total of 86,996 unique peptides (105,811 peptide precursors) corresponding to 6,552 canonical Swiss‐Prot proteins by our new DIA‐MS platform with 2‐h measurement per sample (Mehnert et al , ). This result represents 156 and 51% increases of peptide and protein numbers, compared to the previous SWATH‐MS results acquired on an earlier instrument (Liu et al , ).…”

“…Furthermore, in that particular report, the authors did not use any sample‐specific proteomic database derived from the corresponding RNA‐Seq data to improve AS isoform detection (Zecha et al , ). Herein, we present an integrative analysis comprising of (i) a sensitive and reproducible data‐independent acquisition mass spectrometry (DIA‐MS) measurement (Gillet et al , ; Bruderer et al , ; Amon et al , ; Mehnert et al , ), (ii) an optimized pSILAC‐DIA workflow, (iii) a sample‐specific RNA‐Seq‐derived protein sequence database, and (iv) a novel transcript abundance directed strategy for calling and quantifying AS proteoforms. We then analyzed the protein turnover of differential AS groups across multiple HeLa cell lines collected from different laboratories (Liu et al , ).…”

Global impact of phosphorylation on protein endurance

“…Using DIA-MS 30 , we confidently quantified 24,119 ± 446 Class-I phosphopeptides (with confident phosphorylation site-localization) 4, 31 across 12 HeLa cell lines ( Fig. S1 & Supplementary Table 1).…”

“…For each proteomic and phosphoproteomic sample generated by DeltaSILAC, DIA-MS analysis was performed on 1 μg of peptides, as described previously 30,48 .…”