Cysteinyl Peptide Capture for Shotgun Proteomics: Global Assessment of Chemoselective Fractionation

The complexity of cell and tissue proteomes presents one of the most significant technical challenges in proteomic biomarker discovery. Multidimensional liquid chromatography−tandem mass spectrometry (LC−MS/MS)-based shotgun proteomics can be coupled with selective enrichment of cysteinyl peptides (Cys-peptides) to reduce sample complexity and increase proteome coverage. Here we evaluated the impact of Cys-peptide enrichment on global proteomic inventories. We employed a new cleavable thiol-reactive biotinylating probe, <i>N</i>-(2-(2-(2-(2-(3-(1-hydroxy-2-oxo-2-phenylethyl)phenoxy)acetamido)ethoxy)-ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (IBB), to capture Cys-peptides after digestion. Treatment of tryptic digests with the IBB reagent followed by streptavidin capture and mild alkaline hydrolysis releases a highly purified population of Cys-peptides with a residual <i>S</i>-carboxymethyl tag. Isoelectric focusing (IEF) followed by LC−MS/MS of Cys-peptides significantly expanded proteome coverage in <i>Saccharomyces cerevisiae</i> (yeast) and in human colon carcinoma RKO cells. IBB-based fractionation enhanced detection of Cys-proteins in direct proportion to their cysteine content. The degree of enrichment typically was 2−8-fold but ranged up to almost 20-fold for a few proteins. Published copy number annotation for the yeast proteome enabled benchmarking of MS/MS spectral count data to yeast protein abundance and revealed selective enrichment of cysteine-rich, lower abundance proteins. Spectral count data further established this relationship in RKO cells. Enhanced detection of low abundance proteins was due to the chemoselectivity of Cys-peptide capture, rather than simplification of the peptide mixture through fractionation

Comparison of Protein Immunoprecipitation-Multiple Reaction Monitoring with ELISA for Assay of Biomarker Candidates in Plasma

Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (<i>r</i> = 0.978–0.995) between 10 and 640 ng/mL for the target proteins spiked into a “mock plasma” matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (<i>r</i> = 0.67–0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids

Specificity of the polyclonal antiserum mBicc1p for Bicc1.

<p>(A) The mBicc1-GST antigen was subjected to western blot analysis with the antibodies shown. The anti-Bicc1 (mBicc1p) and anti-GST antibodies, but not the preimmune serum (Pre-IM), recognized the mBicc1-GST antigen of the expected size (43 kD). (B) Schematic representation of the <i>Bicc1</i>-pCMV-Tag4 expression vector constructed by inserting the full-length ORF of <i>Bicc1</i> into Flag-tagged pCMV-Tag4. (C) <i>Bicc1</i>-pCMV-Tag4-transfected HEK293 cell lysates were subjected to western blot analysis with the antibodies shown. The anti-Bicc1 (mBicc1p) and anti-GST antibodies but not the Pre-IM recognized the <i>Bicc1</i>-pCMV-Tag4 protein of the expected size (∼110 kD). (D) Western analysis of duplicated protein lysates from wildtype, <i>Bicc1</i>-silenced (IMCD^shC4C), and <i>Bicc1</i>-overexpressed IMCD cells (IMCD^Bicc1) were blotted with the mBicc1p antibody. Compared to the wildtype control, the immunoreactivity was significantly increased in <i>Bicc1</i>-overexpressed IMCD cells and was almost not detected in the <i>Bicc1</i>-silenced cells. (E) Normalized quantitative analysis of the densitometry values of the western analyses. Compared to wildtype IMCD and <i>Bicc1</i>-silenced IMCD (IMCD^shC4C) cells, <i>Bicc1</i>-overexpressed IMCD (IMCD^Bicc1) cells showed significantly increased Bicc1 expression (*P<0.05). (F) Immunohistochemistry (IHC) staining of Bicc1 protein in the kidneys of E18.5 <i>Bicc1</i>^−/− and its wildtype littermates. Positive staining (arrows) were showed in the wildtype kidney (a), while no obvious positive staining was seen in the E18.5 <i>Bicc1</i>^−/− kidney (b). (a'–b') Corresponding areas of a–b were stained by Bicc1p IHC without counterstaining. Data presented are representative of two to three independently replicated experiments. “cy” = renal cysts. Bars: 25 µm in F.</p

Loss of PC1 downregulates Bicc1 expression <i>in vitro</i>.

<p>(A) qPCR analysis of cultured cell lines with or without <i>Pkd1</i>, <i>Pkd2</i>, or <i>Pkhd1</i> showed no change in the <i>Pkd2</i> or <i>Pkhd1</i> mRNA expression, while <i>Bicc1</i> was significantly downregulated in the cells lacking <i>Pkd1</i> compared to the wildtype control (*P<0.05) (n = 3). (B) Western blot analysis of protein lysates from null-<i>Pkd2</i> cells (E8) and <i>Pkd2</i>-heterozygous cells (D3) using the mBicc1p antibody suggested that PC2 loss did not affect Bicc1 expression. (C) Similar western blots for null-<i>Pkhd1</i> cells (M10H2) and <i>Pkhd1</i>-wildtype cells (W10B2) showed that loss of <i>Pkhd1</i> also did not affect Bicc1 expression. (D) Western blot analysis for the null-<i>Pkd1</i> cells and their wildtype littermate cells showed that loss of <i>Pkd1</i> markedly downregulated the Bicc1 protein expression. (E) Normalized quantitative analysis of the densitometry values of the western analyses. Compared to null-<i>Pkd2</i> or -<i>Pkhd1</i> cells, only the null-<i>Pkd1</i> cells showed significantly reduced Bicc1 expression (*P<0.05). (F) With the wildtype littermate cell control, western blot analyses for the null-<i>Pkd1</i> pool cells and their PC1-CT transfected cells showed that the restoration of PC1 COOH-terminus markedly rescued the downregulation level of Bicc1 expression in the null-<i>Pkd1</i> cells. (G) Normalized quantitative analysis of the densitometry values of the western analyses. Compared to the null-<i>Pkd1</i> cells, the cells with PC1-CT transfection can partially restored Bicc1 expression in the null-<i>Pkd1</i> cells (*P<0.05). Data presented are representative of two to three independently replicated experiments.</p

Bicc1 expression in renal nephrons.

<p>Double immunofluorescence staining with mBicc1p and nephronic markers (LTL, THG, and AQP2) in paraffin-embedded sections of adult wildtype mouse kidneys (n≥5). (A) The Pre-IM was used as a negative staining control. (B) Bicc1 (red) was co-expressed with LTL (green) in the epithelial cells of the renal proximal tubules; (C) Bicc1 (red) was also co-expressed with THG (green) in the epithelial cells of the Henle's thick ascending limbs; (D) With mBicc1p staining (red), Bicc1 was highly expressed in the renal proximal tubules (top arrow) and weakly expressed in the collecting ducts (lower arrow). mBicc1p co-staining with the anti-AQP2 antibody (green) showed weakly co-expression of Bicc1 and AQP2 in the epithelial cells of the collecting ducts (middle arrow). Bar: 30 µm in A–D.</p

Expressional profiles of Bicc1 protein during embryogenesis and kidney development.

<p>By immunohistochemistry (IHC) staining (n≥3), (A) positive labeling is observed in the neural tube (arrow) at E8.5, * indicates the perineural tube mesenchymal tissue; (B) cardiomyocytes in the myocardial wall (arrows) at E9.5, * indicates the pericardio-peritoneal canal; (C) the primordial gut (arrow), * indicates the peritoneal cavity at E10.5; (D) immature hepatocytes (arrow) at E10.5; (E) epithelia of the main bronchi (arrows), + indicates the pericardio-peritoneal canal and * indicates the lung bud tissue; (F) the aortic wall (arrow), * indicates the cardinal vein; and (G) early ureteric bud (lower arrow)/mesonephric duct (upper arrow) at E11.5; (H) the renal comma-shaped body (arrows), (I) epithelia of the pancreatic primordium (arrows), * indicates hepatic tissue and (J) the posterior root ganglions at E12.5, * indicate vertebral bodies. Bicc1 appeared in (K) the adrenal cortex (lower arrow) and cortex of the kidney (upper arrow), * indicates the liver at E16.5; (L) olfactory epithelia (arrows) at E16.5, + indicates the primordial turbinate bone and * indicate the nasal cavities. (M) IHC with mBicc1p antiserum showed positive labeling (arrow) in the newborn mouse kidney. (N) In the 1-month-old kidney, positive staining (arrow) was observed in the juxta-medullary region of the kidney. (O) In the 3-month-old kidney, clear-cut Bicc1 expression (arrow) was seen a similar region of the kidney, but some positive labeling also appeared over the cortical region. (P) Besides of the juxta-medullary and cortical regions, positive labeling also extended to the medullary regions in the 6-month-old kidney. All high-power positive images were from the arrow-pointed regions and placed on the left-lower corners of M–O. (M'–P') showed IHC labeling without counterstaining for the corresponding regions of M–O, respectively. Bars: 10 µm in D; 20 µm in A, C, G–H; 25 µm in E and I; 50 µm in B, F, J–L; 100 µm in M; 150 µm in N–O; 300 µm in P.</p

Loss of PC1 downregulates Bicc1 expression <i>in vivo</i>.

<p>(A) Positive mBicc1p IHC staining (arrows) in the mouse embryonic kidneys. The E14.5 wildtype (a) and its littermate <i>Pkd1</i>^−/− (b) kidneys were stained by mBicc1p antibody (n≥3). In comparisons of kidney tissues with and without <i>Pkd1</i>, the null-<i>Pkd1</i> kidney showed significantly reduced Bicc1 expression. However, there are no Bicc1 expressional difference between the age-matched wildtype (c and e) and their littermate <i>Pkhd1</i>^−/− (d) or littermate <i>Pkd2</i>^−/− (f) kidneys, respectively. “cy” = renal cysts; “G” = glomerulus. (B) Compared to its wildtype littermate, western blot of duplicated tissue lysates from E14.5 embryos showed that loss of PC1 markedly downregulates Bicc1 protein level (left panel). A similar western blot showed equal immunoreactivities between the tissue lysates from E14.5 <i>Pkd2</i>^−/− and its wildtype littermate (middle panel) and between the E14.5 <i>Pkhd1^−/−</i> and its littermate wildtype embryos (n≥3). β-actin for protein loading control. (C) Normalized quantitative analysis of densitometry values of the western analyses. Compared tissues with and without <i>Pkd1</i>, <i>Pkd2</i> or <i>Pkhd1</i>, only null-<i>Pkd1</i> tissue shows significantly reduced Bicc1 expression (*P<0.05). Bars: 50 µm in A.</p

Potency against intracellular <i>T</i>. <i>cruzi</i> amastigotes, VERO host cells and HepG2 cells.

Potency against intracellular T. cruzi amastigotes, VERO host cells and HepG2 cells.</p

RF LAPTc hits.xlsx: Structures, compounds identifier and percent inhibition from primary screen for the 30 hits.

RF LAPTc hits.xlsx: Structures, compounds identifier and percent inhibition from primary screen for the 30 hits.</p

Structures of previously described M17 LAP inhibitors [14,16,21,22,27–31].

Structures of previously described M17 LAP inhibitors [14,16,21,22,27–31].</p