Bhavinkumar B. Patel

Proteomic Analyses of Pancreatic Cyst Fluids

Objectives: There are currently no diagnostic indicators that are consistently reliable, obtainable, and conclusive for diagnosing and risk-stratifying pancreatic cysts. Proteomic analyses were performed to explore pancreatic cyst fluids to yield effective diagnostic biomarkers. Methods: We have prospectively recruited 20 research participants and prepared their pancreatic cyst fluids specifically for proteomic analyses. Proteomic approaches applied were as follows: (1) matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry peptidomics with LC/MS/MS (HPLC-tandem mass spectrometry) protein identification; (2) 2-dimensional gel electrophoresis; (3) GeLC/MS/MS (tryptic digestion of proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by LC/MS/MS). Results: Sequencing of more than 350 free peptides showed that exopeptidase activities rendered peptidomics of cyst fluids unreliable; protein nicking by proteases in the cyst fluids produced hundreds of protein spots from the major proteins, making 2-dimensional gel proteomics unmanageable; GeLC/MS/MS revealed a panel of potential biomarker proteins that correlated with carcinoembryonic antigen (CEA). Conclusions: Two homologs of amylase, solubilized molecules of 4 mucins, 4 solubilized CEA-related cell adhesion molecules (CEACAMs), and 4 S100 homologs may be candidate biomarkers to facilitate future pancreatic cyst diagnosis and risk-stratification. This approach required less than 40 &mgr;L of cyst fluid per sample, offering the possibility to analyze cysts smaller than 1 cm in diameter.

Immunodepletion Plasma Proteomics by TripleTOF 5600 and Orbitrap Elite/LTQ-Orbitrap Velos/Q Exactive Mass Spectrometers

Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.

Assessment of Two Immunodepletion Methods: Off-Target Effects and Variations in Immunodepletion Efficiency May Confound Plasma Proteomics

Immunodepletion of abundant plasma proteins increases the depth of proteome penetration by mass spectrometry. However, the nature and extent of immunodepletion and the effect of off-target depletion on the quantitative comparison of the residual proteins have not been critically addressed. We performed mass spectrometry label-free quantitation to determine which proteins were immunodepleted and by how much. Two immunodepletion resins were compared: Qproteome (Qiagen) which removes albumin+immunoglobulins and Seppro IgY14+SuperMix (Sigma-Aldrich) which removes 14 target proteins plus a number of unidentified proteins. Plasma collected by P100 proteomic plasma collection tubes (BD) from 20 human subjects was individually immunodepleted to minimize potential variability, prior to pooling. The abundant proteins were quantified better when using only albumin+immunoglobulins removal (Qproteome), while lower abundance proteins were evaluated better using exhaustive immunodepletion (Seppro IgY14+SuperMix). The latter resin removed at least 155 proteins, 38% of the plasma proteome in protein number and 94% of plasma protein in mass. The depth of immunodepletion likely accounts for the effectiveness of this resin in revealing low abundance proteins. However, the more profound immunodepletion achieved with the IgY14+SuperMix may lead to false-positive fold-changes between comparison groups if the reproducibility and efficiency of the depletion of a given protein are not considered.

One-Hit Effects in Cancer: Altered Proteome of Morphologically Normal Colon Crypts in Familial Adenomatous Polyposis

We studied patients with Familial Adenomatous Polyposis (FAP) because they are virtually certain to develop colon cancer, and because much is known about the causative APC gene. We hypothesized that the inherited heterozygous mutation itself leads to changes in the proteome of morphologically normal crypts and the proteins that changed may represent targets for preventive and therapeutic agents. We determined the differential protein expression of morphologically normal colon crypts of FAP patients versus those of individuals without the mutation, using two-dimensional gel electrophoresis, mass spectrometry, and validation by two-dimensional gel Western blotting. Approximately 13% of 1,695 identified proteins were abnormally expressed in the morphologically normal crypts of APC mutation carriers, indicating that a colon crypt cell under the one-hit state is already abnormal. Many of the expression changes affect pathways consistent with the function of the APC protein, including apoptosis, cell adhesion, cell motility, cytoskeletal organization and biogenesis, mitosis, transcription, and oxidative stress response. Thus, heterozygosity for a mutant APC tumor suppressor gene alters the proteome of normal-appearing crypt cells in a gene-specific manner, consistent with a detectable one-hit event. These changes may represent the earliest biomarkers of colorectal cancer development, potentially leading to the identification of molecular targets for cancer prevention.

Potential prognostic biomarkers of pancreatic cancer.

Objectives We evaluated whether pancreatic main duct fluid can provide protein biomarkers with prognostic value. Methods Mass spectrometry proteomics was applied to as little as 20µL of fluid collected at the time of tumor surgical resection. Biomarker proteins identified for 27 patients were correlated with clinical outcomes. Results Thirteen patients had pancreatic ductal adenocarcinomas, 4 had intraductal papillary mucinous neoplasm with in situ adenocarcinoma, 5 had ampullary adenocarcinomas, 2 had intraductal papillary mucinous neoplasms, and 3 had benign diseases. In pathologic stage II or higher pancreatic ductal adenocarcinoma, moderate or high expression of S100A8 or S100A9 proteins was associated with a median disease recurrence–free survival of 5.8 months compared with 17.3 months in patients with low expression (P = 0.002). Median overall survival was 12.6 versus 27 months for patients with moderate to high versus low S100A8 and A9 expression (P = 0.02). Conclusions This analysis suggests distinct proteomic signatures for pancreatic cancer. Patients in our study with elevated levels of S100A8 or A9 in the ductal fluid, a near absence of pancreatic enzymes, and high levels of mucins were found to have significantly worse prognosis. Although further validation is needed to corroborate these findings, analysis of pancreatic ductal fluid is a promising tool for identifying biomarkers of interest.

Isobaric Labeling and Data Normalization without Requiring Protein Quantitation

Isobaric multiplexed quantitative proteomics can complement high-resolution sample isolation techniques. Here, we report a simple workflow exponentially modified protein abundance index (emPAI)-MW deconvolution (EMMOL) for normalizing isobaric reporter ratios within and between experiments, where small or unknown amounts of protein are used. EMMOL deconvolutes the isobaric tags for relative and absolute quantification (iTRAQ) data to yield the quantity of each protein of each sample in the pool, a new approach that enables the comparison of many samples without including a channel of reference standard. Moreover, EMMOL allows using a sufficient quantity of control sample to facilitate the peptide fractionation (isoelectric-focusing was used in this report), and mass spectrometry MS/MS sequencing yet relies on the broad dynamic range of iTRAQ quantitation to compare relative protein abundance. We demonstrated EMMOL by comparing four pooled samples with 20-fold range differences in protein abundance and performed data normalization without using prior knowledge of the amounts of proteins in each sample, simulating an iTRAQ experiment without protein quantitation prior to labeling. We used emPAI, the target protein MW, and the iTRAQ reporter ratios to calculate the amount of each protein in each of the four channels. Importantly, the EMMOL-delineated proteomes from separate iTRAQ experiments can be assorted for comparison without using a reference sample. We observed no compression of expression in iTRAQ ratios over a 20-fold range for all protein abundances. To complement this ability to analyze minute samples, we report an optimized iTRAQ labeling protocol for using 5 μg protein as the starting material.

Abstract 1837: Quantitative analysis of AKT/mTOR pathway using immunoprecipitation and targeted mass spectrometry

Background: PI3K/AKT/mTOR pathway plays a central role in tumor progression and anti-cancer drug resistance. The quantitative measurement of protein expression and PTM status of AKT/mTOR pathway proteins is necessary for precise characterization of the disease, monitor cancer progression and determine treatment response. A major bottleneck in the quantitation of AKT/mTOR pathway proteins is the lack of rigorously validated methods/reagents and a reliance on semi-quantitative results from Western blotting. Mass Spectrometry (MS) is increasingly becoming the detection methodology of choice for protein abundance and modifications. Immunoprecipitation (IP) is commonly used upstream of MS as an enrichment tool for low-abundant protein targets. In addition to protein identification, IP can be combined with targeted MS to identify proteins of interest and protein-protein interactions. Here, we used optimized IP-MS to enrich multiple AKT/mTOR pathway proteins simultaneously for targeted selected reaction monitoring (SRM)-based quantitation of protein levels in two human carcinoma cell lines. Methods: A549 and HCT116 cells were stimulated with EGF or IGF. Several AKT/mTOR pathway targets were enriched by improved IP using Protein A/G and Streptavidin magnetic beads and IP eluates were processed using in-solution digestion for LC-MS analysis. A targeted SRM MS assay was developed for quantitation of AKT/mTOR pathway target peptides (EGFR, AKT2, AKT1, PTEN, PIK3CA, and PIK3R1). Multiple targets were also immunoprecipitated simultaneously and quantitated by targeted SRM assay. Improved IP combined with targeted MS workflow was applied to assess recovery of recombinant proteins in a human plasma matrix. Results: Immunoprecipitation resulted in overall higher yield of target protein and less non-specific binding compared to unenriched samples. This enabled us to combine multiple target antibodies to simultaneously enrich multiple pathway protein targets. Enrichment of total and phosphorylated forms of EGFR, AKT isoforms, PI3K and PTEN resulted in quantitation of low to sub nanogram levels of targets in two cell lysates by targeted MS (LC-SRM/MS). In addition, IP coupled with targeted MS was used to enrich and quantify as low as 10ng/mL recombinant EGFR, AKT2/AKT1, PTEN and PI3K spiked into a human plasma matrix. Conclusion: IP combined with targeted MS permits absolute quantitation of AKT/mTOR pathway proteins and PTMs at low to sub nanogram concentrations. This multiplex targeted assay can be used for verification and validation of AKT/mTOR pathway proteins in other cancer cell lines or tissue samples. Citation Format: Bhavinkumar Patel, Suzanne Smith, Ryan Bomgarden, Kay Opperman, Barbara Kaboord, John Rogers. Quantitative analysis of AKT/mTOR pathway using immunoprecipitation and targeted mass spectrometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1837. doi:10.1158/1538-7445.AM2015-1837