PMID- 32419461
OWN - NLM
STAT- MEDLINE
DCOM- 20210617
LR  - 20210808
IS  - 1535-3907 (Electronic)
IS  - 1535-3893 (Print)
IS  - 1535-3893 (Linking)
VI  - 19
IP  - 8
DP  - 2020 Aug 7
TI  - Toward a Universal Sample Preparation Method for Denaturing Top-Down Proteomics 
      of Complex Proteomes.
PG  - 3315-3325
LID - 10.1021/acs.jproteome.0c00226 [doi]
AB  - A universal and standardized sample preparation method becomes vital for 
      denaturing top-down proteomics (dTDP) to advance the scale and accuracy of 
      proteoform delineation in complex biological systems. It needs to have high 
      protein recovery, minimum bias, good reproducibility, and compatibility with 
      downstream mass spectrometry (MS) analysis. Here, we employed a lysis buffer 
      containing sodium dodecyl sulfate for extracting proteoforms from cells and, for 
      the first time, compared membrane ultrafiltration (MU), chloroform-methanol 
      precipitation (CMP), and single-spot solid-phase sample preparation using 
      magnetic beads (SP3) for proteoform cleanup for dTDP. The MU method outperformed 
      CMP and SP3 methods, resulting in high and reproducible protein recovery from 
      both Escherichia coli cell (59 +/- 3%) and human HepG2 cell (86 +/- 5%) samples 
      without a significant bias. Single-shot capillary zone electrophoresis 
      (CZE)-MS/MS analyses of the prepared E. coli and HepG2 cell samples using the MU 
      method identified 821 and 516 proteoforms, respectively. Nearly 30 and 50% of the 
      identified E. coli and HepG2 proteins are membrane proteins. CZE-MS/MS identified 
      94 histone proteoforms from the HepG2 sample with various post-translational 
      modifications, including acetylation, methylation, and phosphorylation. Our 
      results suggest that combining the SDS-based protein extraction and the MU-based 
      protein cleanup could be a universal sample preparation method for dTDP. The MS 
      raw data have been deposited to the ProteomeXchange Consortium with the data set 
      identifier PXD018248.
FAU - Yang, Zhichang
AU  - Yang Z
AD  - Department of Chemistry, Michigan State University, 578 S Shaw Ln, East Lansing, 
      Michigan 48824 United States.
FAU - Shen, Xiaojing
AU  - Shen X
AUID- ORCID: 0000-0003-2079-9115
AD  - Department of Chemistry, Michigan State University, 578 S Shaw Ln, East Lansing, 
      Michigan 48824 United States.
FAU - Chen, Daoyang
AU  - Chen D
AD  - Department of Chemistry, Michigan State University, 578 S Shaw Ln, East Lansing, 
      Michigan 48824 United States.
FAU - Sun, Liangliang
AU  - Sun L
AUID- ORCID: 0000-0001-8939-5042
AD  - Department of Chemistry, Michigan State University, 578 S Shaw Ln, East Lansing, 
      Michigan 48824 United States.
LA  - eng
GR  - R01 GM125991/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20200529
PL  - United States
TA  - J Proteome Res
JT  - Journal of proteome research
JID - 101128775
RN  - 0 (Escherichia coli Proteins)
RN  - 0 (Proteome)
SB  - IM
MH  - Escherichia coli/genetics
MH  - *Escherichia coli Proteins
MH  - Humans
MH  - *Proteome
MH  - Proteomics
MH  - Reproducibility of Results
MH  - Tandem Mass Spectrometry
PMC - PMC7542658
MID - NIHMS1634043
OTO - NOTNLM
OT  - CZE-MS/MS
OT  - PTMs
OT  - SDS
OT  - SP3
OT  - chloroform-methanol precipitation
OT  - denaturing top-down proteomics
OT  - histone
OT  - membrane proteins
OT  - membrane ultrafiltration
OT  - sample preparation
COIS- The authors declare no competing financial interest.
EDAT- 2020/05/19 06:00
MHDA- 2021/06/22 06:00
PMCR- 2021/08/07
CRDT- 2020/05/19 06:00
PHST- 2020/05/19 06:00 [pubmed]
PHST- 2021/06/22 06:00 [medline]
PHST- 2020/05/19 06:00 [entrez]
PHST- 2021/08/07 00:00 [pmc-release]
AID - 10.1021/acs.jproteome.0c00226 [doi]
PST - ppublish
SO  - J Proteome Res. 2020 Aug 7;19(8):3315-3325. doi: 10.1021/acs.jproteome.0c00226. 
      Epub 2020 May 29.