5 research outputs found
Searching Missing Proteins Based on the Optimization of Membrane Protein Enrichment and Digestion Process
A membrane protein
enrichment method composed of ultracentrifugation
and detergent-based extraction was first developed based on MCF7 cell
line. Then, in-solution digestion with detergents and eFASP (enhanced
filter-aided sample preparation) with detergents were compared with
the time-consuming in-gel digestion method. Among the in-solution
digestion strategies, the eFASP combined with RapiGest identified
1125 membrane proteins. Similarly, the eFASP combined with sodium
deoxycholate identified 1069 membrane proteins; however, the in-gel
digestion characterized 1091 membrane proteins. Totally, with the
five digestion methods, 1390 membrane proteins were identified with
≥1 unique peptides, among which 1345 membrane proteins contain
unique peptides ≥2. This is the biggest membrane protein data
set for MCF7 cell line and even breast cancer tissue samples. Interestingly,
we identified 13 unique peptides belonging to 8 missing proteins (MPs).
Finally, eight unique peptides were validated by synthesized peptides.
Two proteins were confirmed as MPs, and another two proteins were
candidate detections
iTRAQ-Based Membrane Proteomics Reveals Plasma Membrane Proteins Change During HepaRG Cell Differentiation
HepaRG cell, a stabilized bipotent
liver progenitor cell line,
exhibits hepatocyte functions only after differentiation. However,
the mechanism of transition from nondifferentiated to differentiated
states, accompanied by proliferation migration and differentiation,
remains poorly understood, particularly those proteins residing in
the plasma membrane. In this study, the membrane protein expression
change of HepaRG cell during differentiation were systematically analyzed
using an iTRAQ labeled quantitative membrane proteomics approach.
A total of 70 membrane proteins were identified to be differentially
expressed among 849 quantified membrane proteins. Function and disease
clustering analysis proved that 11 of these proteins are involved
in proliferation, migration, and differentiation. Two key factors
(MMP-14 and OCLN) were validated by qRT-PCR and Western blot. Blockade
of MMP-14 further demonstrated its important function during tumor
cell migration. The data sets have been uploaded to ProteomeXchange
with the identifier PXD004752
Deep Coverage Proteomics Identifies More Low-Abundance Missing Proteins in Human Testis Tissue with Q‑Exactive HF Mass Spectrometer
Since 2012, missing proteins (MPs)
investigation has been one of
the critical missions of Chromosome-Centric Human Proteome Project
(C-HPP) through various biochemical strategies. On the basis of our
previous testis MPs study, faster scanning and higher resolution mass-spectrometry-based
proteomics might be conducive to MPs exploration, especially for low-abundance
proteins. In this study, Q-Exactive HF (HF) was used to survey proteins
from the same testis tissues separated by two separating methods (tricine-
and glycine-SDS-PAGE), as previously described. A total of 8526 proteins
were identified, of which more low-abundance proteins were uniquely
detected in HF data but not in our previous LTQ Orbitrap Velos (Velos)
reanalysis data. Further transcriptomics analysis showed that these
uniquely identified proteins by HF also had lower expression at the
mRNA level. Of the 81 total identified MPs, 74 and 39 proteins were
listed as MPs in HF and Velos data sets, respectively. Among the above
MPs, 47 proteins (43 neXtProt PE2 and 4 PE3) were ranked as confirmed
MPs after verifying with the stringent spectra match and isobaric
and single amino acid variants filtering. Functional investigation
of these 47 MPs revealed that 11 MPs were testis-specific proteins
and 7 MPs were involved in spermatogenesis process. Therefore, we
concluded that higher scanning speed and resolution of HF might be
factors for improving the low-abundance MP identification in future
C-HPP studies. All mass-spectrometry data from this study have been
deposited in the ProteomeXchange with identifier PXD004092
Special Enrichment Strategies Greatly Increase the Efficiency of Missing Proteins Identification from Regular Proteome Samples
As part of the Chromosome-Centric
Human Proteome Project (C-HPP)
mission, laboratories all over the world have tried to map the entire
missing proteins (MPs) since 2012. On the basis of the first and second
Chinese Chromosome Proteome Database (CCPD 1.0 and 2.0) studies, we
developed systematic enrichment strategies to identify MPs that fell
into four classes: (1) low molecular weight (LMW) proteins, (2) membrane
proteins, (3) proteins that contained various post-translational modifications
(PTMs), and (4) nucleic acid-associated proteins. Of 8845 proteins
identified in 7 data sets, 79 proteins were classified as MPs. Among
data sets derived from different enrichment strategies, data sets
for LMW and PTM yielded the most novel MPs. In addition, we found
that some MPs were identified in multiple-data sets, which implied
that tandem enrichments methods might improve the ability to identify
MPs. Moreover, low expression at the transcription level was the major
cause of the “missing” of these MPs; however, MPs with
higher expression level also evaded identification, most likely due
to other characteristics such as LMW, high hydrophobicity and PTM.
By combining a stringent manual check of the MS<sub>2</sub> spectra
with peptides synthesis verification, we confirmed 30 MPs (neXtProt
PE2 ∼ PE4) and 6 potential MPs (neXtProt PE5) with authentic
MS evidence. By integrating our large-scale data sets of CCPD 2.0,
the number of identified proteins has increased considerably beyond
simulation saturation. Here, we show that special enrichment strategies
can break through the data saturation bottleneck, which could increase
the efficiency of MP identification in future C-HPP studies. All 7
data sets have been uploaded to ProteomeXchange with the identifier
PXD002255
Tissue-Based Proteogenomics Reveals that Human Testis Endows Plentiful Missing Proteins
Investigations
of missing proteins (MPs) are being endorsed by
many bioanalytical strategies. We proposed that proteogenomics of
testis tissue was a feasible approach to identify more MPs because
testis tissues have higher gene expression levels. Here we combined
proteomics and transcriptomics to survey gene expression in human
testis tissues from three post-mortem individuals. Proteins were extracted
and separated with glycine- and tricine-SDS-PAGE. A total of 9597
protein groups were identified; of these, 166 protein groups were
listed as MPs, including 138 groups (83.1%) with transcriptional evidence.
A total of 2948 proteins are designated as MPs, and 5.6% of these
were identified in this study. The high incidence of MPs in testis
tissue indicates that this is a rich resource for MPs. Functional
category analysis revealed that the biological processes that testis
MPs are mainly involved in are sexual reproduction and spermatogenesis.
Some of the MPs are potentially involved in tumorgenesis in other
tissues. Therefore, this proteogenomics analysis of individual testis
tissues provides convincing evidence of the discovery of MPs. All
mass spectrometry data from this study have been deposited in the
ProteomeXchange (data set identifier PXD002179)