5 research outputs found
Transcription Factor Response Elements on Tip: A Sensitive Approach for Large-Scale Endogenous Transcription Factor Quantitative Identification
The ability to map endogenous transcription
factors (TFs) DNA binding activity at the proteome scale will greatly
enhance our understanding of various biological processes. Here we
report a highly sensitive, rapid, and high-throughput approach, transcription
factor response elements on tip-mass spectrometry (TOT-MS), that allows
for quantitative measurement of endogenous TFs. A total of 150 TFs
from 1 Ī¼g of nuclear extracts can be quantified with single
shot mass spectrometry detection in 1 h of machine time. Up to 755
TFs, which is comparable to the depth of RNA-seq, were identified
by TOT coupled with on-tip small size reverse-phase liquid chromatography.
We further demonstrated the capability of TOT-MS by interrogating
the dynamic change of TFs in the epidermal growth factor (EGF) signaling
pathway. This approach should find broad applications in elucidating
the TF landscape from limited amounts of biological materials
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 āmissingā 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 ā¼ 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)
First Proteomic Exploration of Protein-Encoding Genes on Chromosome 1 in Human Liver, Stomach, and Colon
The launch of the Chromosome-Centric Human Proteome Project
provides an opportunity to gain insight into the human proteome. The
Chinese Human Chromosome Proteome Consortium has initiated proteomic
exploration of protein-encoding genes on human chromosomes 1, 8, and
20. Collaboration within the consortium has generated a comprehensive
proteome data set using normal and carcinomatous tissues from human
liver, stomach, and colon and 13 cell lines originating in these organs.
We identified 12,101 proteins (59.8% coverage against Swiss-Prot human
entries) with a protein false discovery rate of less than 1%. On chromosome
1, 1,252 proteins mapping to 1,227 genes, representing 60.9% of Swiss-Prot
entries, were identified; however, 805 proteins remain unidentified,
suggesting that analysis of more diverse samples using more advanced
proteomic technologies is required. Genes encoding the unidentified
proteins were concentrated in seven blocks, located at p36, q12-21,
and q42-44, partly consistent with correlation of these blocks with
cancers of the liver, stomach, and colon. Combined transcriptome,
proteome, and cofunctionality analyses confirmed 23 coexpression clusters
containing 165 genes. Biological information, including chromosome
structure, GC content, and protein coexpression pattern was analyzed
using multilayered, circular visualization and tabular visualization.
Details of data analysis and updates are available in the Chinese
Chromosome-Centric Human Proteome Database (http://proteomeview.hupo.org.cn/chromosome/)
First Proteomic Exploration of Protein-Encoding Genes on Chromosome 1 in Human Liver, Stomach, and Colon
The launch of the Chromosome-Centric Human Proteome Project
provides an opportunity to gain insight into the human proteome. The
Chinese Human Chromosome Proteome Consortium has initiated proteomic
exploration of protein-encoding genes on human chromosomes 1, 8, and
20. Collaboration within the consortium has generated a comprehensive
proteome data set using normal and carcinomatous tissues from human
liver, stomach, and colon and 13 cell lines originating in these organs.
We identified 12,101 proteins (59.8% coverage against Swiss-Prot human
entries) with a protein false discovery rate of less than 1%. On chromosome
1, 1,252 proteins mapping to 1,227 genes, representing 60.9% of Swiss-Prot
entries, were identified; however, 805 proteins remain unidentified,
suggesting that analysis of more diverse samples using more advanced
proteomic technologies is required. Genes encoding the unidentified
proteins were concentrated in seven blocks, located at p36, q12-21,
and q42-44, partly consistent with correlation of these blocks with
cancers of the liver, stomach, and colon. Combined transcriptome,
proteome, and cofunctionality analyses confirmed 23 coexpression clusters
containing 165 genes. Biological information, including chromosome
structure, GC content, and protein coexpression pattern was analyzed
using multilayered, circular visualization and tabular visualization.
Details of data analysis and updates are available in the Chinese
Chromosome-Centric Human Proteome Database (http://proteomeview.hupo.org.cn/chromosome/)