TiO₂ with Tandem Fractionation (TAFT): An Approach for Rapid, Deep, Reproducible, and High-Throughput Phosphoproteome Analysis

Mass-spectrometry-based phosphoproteomic workflows traditionally require efficient prefractionation and enrichment of phosphopeptides to gain an in-depth, global, and unbiased systematic investigation of phosphoproteome. Here we present TiO₂ with tandem fractionation (TAFT) approach, which combines titanium dioxide (TiO₂) enrichment and tandem high-pH reverse-phase (HpRP) for phosphoproteome analysis in a high-throughput manner; the entire workflow takes only 3 h to complete without laborious phosphopeptide preparation. We applied this approach to HeLa and HepG2.2.15 cells to characterize the capability of TAFT approach, which enables deep identification and quantification of more than 14 000 unique phosphopeptides in a single sample from 1 mg of protein as starting materials in <4 h of MS measurement. In total, we identified and quantified 21 281 phosphosites in two cell lines with >91% selectivity and high quantitative reproducibility (average Pearson correlation is 0.90 between biological replicates). More generally, the presented approach enables rapid, deep, and reproducible phosphoproteome analysis in a high-throughput manner with low cost, which should facilitate our understanding of signaling networks in a wide range of biological systems or the process of clinical applications

Discovery of Novel Genes and Gene Isoforms by Integrating Transcriptomic and Proteomic Profiling from Mouse Liver

Comprehensively identifying gene expression in both transcriptomic and proteomic levels of one tissue is a prerequisite for a deeper understanding of its biological functions. Alternative splicing and RNA editing, two main forms of transcriptional processing, play important roles in transcriptome and proteome diversity and result in multiple isoforms for one gene, which are hard to identify by mass spectrometry (MS)-based proteomics approach due to the relative lack of isoform information in standard protein databases. In our study, we employed MS and RNA-Seq in parallel into mouse liver tissue and captured a considerable catalogue of both transcripts and proteins that, respectively, covered 60 and 34% of protein-coding genes in Ensembl. We then developed a bioinformatics workflow for building a customized protein database that for the first time included new splicing-derived peptides and RNA-editing-caused peptide variants, allowing us to more completely identify protein isoforms. Using this experimentally determined database, we totally identified 150 peptides not present in standard biological databases at false discovery rate of <1%, corresponding to 72 novel splicing isoforms, 43 new genetic regions, and 15 RNA-editing sites. Of these, 11 randomly selected novel events passed experimental verification by PCR and Sanger sequencing. New discoveries of gene products with high confidence in two omics levels demonstrated the robustness and effectiveness of our approach and its potential application into improve genome annotation. All the MS data have been deposited to the iProx (http://ww.iprox.org) with the identifier IPX00003601

Discovery of Novel Genes and Gene Isoforms by Integrating Transcriptomic and Proteomic Profiling from Mouse Liver

Comprehensively identifying gene expression in both transcriptomic and proteomic levels of one tissue is a prerequisite for a deeper understanding of its biological functions. Alternative splicing and RNA editing, two main forms of transcriptional processing, play important roles in transcriptome and proteome diversity and result in multiple isoforms for one gene, which are hard to identify by mass spectrometry (MS)-based proteomics approach due to the relative lack of isoform information in standard protein databases. In our study, we employed MS and RNA-Seq in parallel into mouse liver tissue and captured a considerable catalogue of both transcripts and proteins that, respectively, covered 60 and 34% of protein-coding genes in Ensembl. We then developed a bioinformatics workflow for building a customized protein database that for the first time included new splicing-derived peptides and RNA-editing-caused peptide variants, allowing us to more completely identify protein isoforms. Using this experimentally determined database, we totally identified 150 peptides not present in standard biological databases at false discovery rate of <1%, corresponding to 72 novel splicing isoforms, 43 new genetic regions, and 15 RNA-editing sites. Of these, 11 randomly selected novel events passed experimental verification by PCR and Sanger sequencing. New discoveries of gene products with high confidence in two omics levels demonstrated the robustness and effectiveness of our approach and its potential application into improve genome annotation. All the MS data have been deposited to the iProx (http://ww.iprox.org) with the identifier IPX00003601

Relatively Low Level of Antigen-specific Monocytes Detected in Blood from Untreated Tuberculosis Patients Using CD4⁺ T-cell Receptor Tetramers

<div><p>The <em>in vivo</em> kinetics of antigen-presenting cells (APCs) in patients with advanced and convalescent tuberculosis (TB) is not well characterized. In order to target <em>Mycobacterium tuberculosis</em> (MTB) peptides- and HLA-DR-holding monocytes and macrophages, 2 MTB peptide-specific CD4⁺ T-cell receptor (TCR) tetramers eu and hu were successfully constructed. Peripheral blood (PBL) samples from inpatients with advanced pulmonary TB (PTB) were analyzed using flow cytometry, and the percentages of tetramer-bound CD14⁺ monocytes ranged from 0.26–1.44% and 0.21–0.95%, respectively; significantly higher than those measured in PBL samples obtained from non-TB patients, healthy donors, and umbilical cords. These tetramers were also able to specifically detect macrophages <em>in situ via</em> immunofluorescent staining. The results of the continuous time-point tracking of the tetramer-positive rates in PBL samples from active PTB outpatients undergoing treatment show that the median percentages were at first low before treatment, increased to their highest levels during the first month, and then began to decrease during the second month until finally reaching and maintaining a relatively low level after 3–6 months. These results suggest that there is a relatively low level of MTB-specific monocytes in advanced and untreated patients. Further experiments show that MTB induces apoptosis in CD14⁺ cells, and the percentage of apoptotic monocytes dramatically decreases after treatment. Therefore, the relatively low level of MTB-specific monocytes is probably related to the apoptosis or necrosis of APCs due to live bacteria and their growth. The bactericidal effects of anti-TB drugs, as well as other unknown factors, would induce a peak value during the first month of treatment, and a relatively low level would be subsequently reached and maintained until all of the involved factors reached equilibrium. These tetramers have diagnostic potential and can provide valuable insights into the mechanisms of antigen presentation and its relationship with TB infection and latent TB infection.</p> </div

<i>In situ</i> detection of tetramer-bound MTB APCs using confocal laser-scanning microscopy.

<p>(A) The MTB antigen and peptide/HLA-DR were detected using <i>in situ</i> immunofluorescent staining with anti-MTB antibody and TCR tetramer, respectively. The nucleus is stained blue (panel 1), TCR tetramer is stained green (panel 2), and MTB antigen is stained red (panel 3). Panel 4 shows a merged image. (B) CD14 and peptide/HLA-DR were detected using i<i>n situ</i> immunofluorescent staining with anti-CD14 antibody and TCR tetramer, respectively. The nucleus is labeled in blue (panel 1), CD14 is labeled in green (panel 2), and the TCR tetramer is labeled in red (panel 3). Panel 4 shows a merged image. Lymph node and lung sections of active TB patients show co-staining in both (A) and (B), but no staining or only anti-CD14 antibody staining were observed in the lung and lymph node sections from non-PTB patients.</p

HLA-DRB1 alleles of 9 active PTB outpatients.

<p>HLA-DRB1 alleles of 9 active PTB outpatients.</p

Statistical analyses of the tetramer-bound CD14⁺ monocytes detected in PBL samples from active PTB inpatients.

<p>The percentage of tetramer-bound CD14⁺ monocytes was detected by flow cytometry. (A) The clustered bar graph shows that the median percentages of tetramer-bound CD14⁺ monocytes in the PBL samples from PTB inpatients (second bar; 0.6% and 0.45%) were significantly higher than those of the control groups (Mann-Whitney U test, <i>p</i><0.01). N1 and N2 indicate the number of samples stained with the eu- and hu-tetramer in each group, respectively. (B) The scatter graphs show that a high percentage of tetramer-bound CD14⁺ monocytes was only observed in PBL samples from PTB inpatients following staining with both tetramers.</p

HLA-DRB1 alleles of active PTB patients and the percentages of TCR tetramer-bound CD14⁺ monocytes in PBL samples.

a<p>The positivity rate indicates the median percentage of double-stained-positive monocytes in PBL samples from active PTB patients.</p

VDJ repertoire, CDR3 amino acid sequences of the α/β chains, and other related information regarding CD4⁺ TCR eu- and hu-tetramers.

<p>VDJ repertoire, CDR3 amino acid sequences of the α/β chains, and other related information regarding CD4⁺ TCR eu- and hu-tetramers.</p

Percentage of early-apoptosis CD14⁺ cells from healthy donors or the continuously treated PTB patients.

<p>Apoptotic CD14⁺ cells were analyzed by flow cytometry using Annexin V plus PI staining <i>in vitro</i>. Annexin V⁺PI⁻ cells represent the early apoptotic populations. Healthy, healthy donors; untreated, untreated PTB patients; within 5 days, PTB patients who received regular anti-TB treatment within 5 days; 15 to 30 days, PTB patients received regular treatment for 15–30 days; More than 30 days: PTB patients received regular treatment for >30 days. The presented data indicate that the percentage of apoptotic cells dramatically decreased after treatment.</p