Liquid Chromatography-multiple Reaction Monitoring Mass Research Articles

Abstract 2494: Liquid chromatography-multiple reaction monitoring mass spectrometry biomarker quantification for breast cancer patient assessment.

Abstract Introduction: A quantitative mass spectrometry platform has been developed for measuring biomarkers of protein expression and phosphorylation in breast cancer. The quantification of numerous biomarkers from a single tissue specimen has the potential to impact patient care. Specifically, the focus of initial development was on the expression and phosphorylation of estrogen receptor alpha (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). However, immunohistochemical assessments of Ki-67 and vimentin are also widely used for monitoring proliferation in breast cancer. The integration of tissue quality control measurements, quantification of proliferation biomarkers, and ER/PR/HER2 evaluation could improve patient assessment and open novel avenues for patient classification, prognosis, and selection of therapy in breast cancer. Experimental Procedures: Breast cancer cell lines MCF7, BT474, and T47D were used to develop peptide-based quantitative assays, including stable isotope-labeled standard peptides, (n = 31) for protein expression and phosphorylation biomarkers using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) of digested whole cell lysates or SDS-PAGE fractionated proteins followed by immunoprecipitation to examine expression and phosphorylation of ER/PR/HER2 (IP-LC-MRM). Using 2 mg of total protein, the sample is divided into three parts. Tissue QC biomarkers and expression levels of Ki67, vimentin, and HER2 are measured in digests of whole cell lysate or tissue homogenate (1% of sample). Then, parallel IP strategies are used for HER2 (10% of sample) and ER/PR (90%) before LC-MRM analysis of expression and phosphorylation. Multiplex IP of the three proteins was not successful, because the amplification of HER2 could not be accommodated. The sensitivity of each assay was tested using serial dilutions of cell lysate to evaluate requirements for analysis of clinical specimens. Data Summary: LC-MRM assays for both unmodified and phosphorylated peptides have been developed and characterized for each protein using cell line models. Assays have been implemented in cell lines to study Estradiol/EGF stimulation as well as lapatinib inhibition of HER2. Data has been generated for pre-treatment frozen tissue specimens from breast cancer patients that are either HER2+ or ER+ (n = 12 per group). The analysis of a larger group of patients and in clinical scenarios (e.g. comparison of naïve and drug resistant tissues from the same patient) will establish the clinical utility of these assays. Conclusions: The ability to examine protein expression as well as phosphorylation status in these biomarkers with quantitative mass spectrometry has the potential to improve the selection of targeted therapeutics. Citation Format: Yi Chen, David J. Britton, Elizabeth R. Wood, Anthony M. Magliocco, Ian Pike, John M. Koomen. Liquid chromatography-multiple reaction monitoring mass spectrometry biomarker quantification for breast cancer patient assessment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2494. doi:10.1158/1538-7445.AM2013-2494

Abstract 3209: Elucidating drug resistance mechanisms in melanoma with quantitative proteomics.

Abstract Introduction: Exciting breakthroughs have been made in targeted therapy for melanoma; however, the development of drug resistance remains a critical problem for patients due to the speed of onset and the numerous complex mechanisms. Parallel development of platform for quantitative protein expression enables biomarker monitoring to evaluate the multiple pathways through which tumor cells can acquire drug resistance. We have developed quantitative assays using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) to assess a diverse panel of >70 proteins involved in signaling pathways, including apoptosis, receptor tyrosine kinases, PI3K/Akt/MTOR, and MAP kinases. Assays quantify changes in response to targeted therapeutic agents and examine subsequent resistance mechanisms in cell lines, xenografts, and biopsies from patients. Experimental procedures: Naïve and BRAF V600E inhibitor-resistant melanoma cell lines were treated with vehicle control, BRAF V600E inhibitor (PLX4720), HSP90 inhibitor (XL-888, Exelixis), or MEK inhibitor (AZD6244). Immunoblotting and LC-MRM were used to evaluate protein expression levels. Heat shock proteins can be measured directly from whole cell lysates or tumor homogenates using a few micrograms of protein. For expression of cancer signaling proteins (many of which are HSP90 clients), 50 micrograms of total protein were fractionated by SDS-PAGE prior to digestion and LC-MRM quantification. Similar experiments were conducted with mouse xenograft tumors from treated and control mice; these measurements have also been applied to fine needle aspirates (FNAs) and core biopsies from patients in the context of a clinical trial. Data summary: LC-MRM quantification is effective in quantifying these proteins in cell lines, xenografts, and biopsies or FNAs. The LC-MRM panel assay was able to screen for a diverse range of proteins revealing previously undescribed cellular responses to treatment and elucidate potential mechanisms of resistance. As an example, both LC-MRM data and Westerns showed consistent responses to HSP90 inhibition, including increased expression of HSP71 and reduction of HSP90 client levels. Proteins were also modulated in response and resistance to BRAF and MEK inhibitors. Conclusion: LC-MRM quantifies biomarker panels and provides an assessment tool that can translated in a straightforward way from cell line models to patient specimens. This emerging technique holds great promise for biopsy-mandated clinical trials, particularly in diseases like melanoma that can be readily sampled. Measurements using this LC-MRM platform can be used to assess drug performance and patient response to evaluate treatment, detect drug resistance, and suggest rational combination therapies. Citation Format: Elizabeth Remily-Wood, Kim Paraiso, Eirik Haarberg, Y. Ann Chen, Vito Rebecca, Keiran Smalley, John M. Koomen. Elucidating drug resistance mechanisms in melanoma with quantitative proteomics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3209. doi:10.1158/1538-7445.AM2013-3209

A combination of untargeted and targeted metabolomics approaches unveils changes in the kynurenine pathway following cardiopulmonary resuscitation

The mechanisms responsible for post-resuscitation myocardial and cerebral dysfunction are not well understood, especially in the early post-resuscitation phases. In this investigation, we first adopted unbiased mass spectrometry-based metabolomic profiling to identify perturbations in circulating metabolites in a rat model of cardiac arrest and cardiopulmonary resuscitation. Our findings strongly indicated early alterations in a major route of the tryptophan catabolism, namely the kynurenines pathway, after resuscitation. Specific metabolites involved in the tryptophan catabolism were quantified absolutely using liquid chromatography-multiple reaction monitoring-mass spectrometry. Tryptophan plasma concentration fell significantly very early in the post-resuscitation phase, while its metabolites, l-kynurenine, kynurenic acid, 3-hydroxyanthranilic acid and 5-hydroxyindoleacetic acid, rose significantly. Changes in their concentration reflected changes in rat post-resuscitation myocardial dysfunction. Elevated plasma level of kynurenic acid, 3-hydroxyanthranilic acid were associated with significant decrease in ejection fraction and stroke volume. It is well known that kynurenines pathway is involved in the pathogenesis of numerous central nervous system disorders. By implication, altered levels of tryptophan metabolites in the early post resuscitation phase might contribute to the degree of cognitive recovery. Our results suggest that kynurenine pathway is activated early following resuscitation from cardiac arrest and might account for the severity of post-resuscitation syndrome. Our explorative investigation indicate that metabolomics can help to clarify unexplored biochemical pathways in cardiopulmonary resuscitation.

Quantifying the Cluster of Differentiation 4 Receptor Density on Human T Lymphocytes Using Multiple Reaction Monitoring Mass Spectrometry

Cluster of differentiation 4 (CD4) is an important glycoprotein containing four extracellular domains, a transmembrane portion and a short intracellular tail. It locates on the surface of various types of immune cells and performs a critical role in multiple cellular functions such as signal amplification and activation of T cells. It is well-known as a clinical cell surface protein marker for study of HIV progression and for defining the T helper cell population in immunological applications. Moreover, CD4 protein has been used as a biological calibrator for quantification of other surface and intracellular proteins. However, flow cytometry, the conventional method of quantification of the CD4 density on the T cell surface depends on antibodies and has suffered from variables such as antibody clones, the fluorophore and conjugation chemistries, the fixation conditions, and the flow cytometric quantification methods used. In this study, we report the development of a highly reproducible nano liquid chromatography-multiple reaction monitoring mass spectrometry-based quantitative method to quantify the CD4 receptor density in units of copy number per cell on human CD4+ T cells. The method utilizes stable isotope-labeled full-length standard CD4 as an internal standard to measure endogenous CD4 directly, without the use of antibodies. The development of the mass spectrometry-based approach of CD4 protein quantification is important as a complementary strategy to validate the analysis from the cytometry-based conventional method. It also provides new support for quantitative understanding and advanced characterization of CD4 on CD4+ T cells.

Abstract 4810: Multiplexed immunoprecipitation-liquid chromatography-multiple reaction monitoring mass spectrometry quantification of protein expression and phosphorylation for breast cancer biomarkers

Abstract Introduction: Estrogen receptor alpha (ER alpha), progesterone receptor B (PR B), and the receptor tyrosine kinase, Her2, are tumor markers classified by the American Society of Clinical Oncology (ASCO) as level of evidence I in breast cancer. The quantitative evaluation of protein expression and phosphorylation status of these targets in tumor tissues will enable subtype classification and support clinical decision-making and selection of targeted therapy in breast cancer. Experimental Procedures: Breast cancer cell lines were used to develop peptide-based quantitative assays for protein expression and phosphorylation using immunoprecipitation (IP) and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM). Stable isotope-labeled standard peptides were synthesized and developed as internal standards. Multiplexed IP for ER alpha, PR B, and HER2 will be developed to enable the enrichment of the proteins to enable quantification of the expression and phosphorylation of these biomarkers in the same sample. The sensitivity of each assay will be tested using serial dilutions of cell lysate to evaluate requirements for analysis of clinical specimens. Data Summary: LC-MRM assays for both unmodified and phosphorylated peptides have been developed and characterized for each protein. Recovery using multiplexed IP for these proteins is similar to recovery of individual IP. Data have been generated for cell line models of stimulation and treatment. For example, LC-MRM quantification of HER2 in cell line models stimulated with EGF or Heregulin shows that protein expression is unchanged, but phosphorylation increases; on the other hand, LC-MRM shows clearly that phosphorylation of Her2 is inhibited with Lapatinib. Conclusions: The ability to examine the protein expression as well as the phosphorylation status in these biomarkers with quantitative mass spectrometry will produce improved understanding of their role in the development and progression of breast cancer and has the potential to improve the selection of targeted therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4810. doi:1538-7445.AM2012-4810

Abstract 4809: Elucidating acquired drug resistance in multiple myeloma using expression proteomics and phosphorylation profiling

Abstract Introduction: Multiple myeloma (MM) is an incurable malignancy of the plasma cell. While patients may initially respond to treatment, the development of acquired drug resistance (ADR) is a consistent feature of the disease. In order to understand the processes involved in ADR, proteomics is used to examine multiple phenotypic outcomes associated with drug resistance in cell line models. Both protein expression and phosphorylation data will be mined for biological mechanisms and candidate biomarkers for detection of ADR in MM patients. Experimental Procedures: Using cell line models of melphalan and doxorubicin resistance, protein expression is compared using spectral counts and extracted ion chromatograms from GeLC-MS/MS data. In addition, phosphorylation profiles are created using SCX peptide fractionation with IMAC phosphopeptide enrichment prior to LC-MS/MS cataloging. Additional hypothesis-driven experiments are conducted using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for targeted quantification. In addition to steady state comparisons between naïve and drug resistant cells, initial responses to therapy are also evaluated. Assays developed for interrogation of cell line models can be translated to patient specimens. Data Summary: GeLC-MS/MS expression measurements typically produce semi-quantitative data for > 2,000 proteins (2+ peptides per protein). Phosphorylation analysis has produced a catalog of > 1,000 sites across the entire group of cell lines. Data are manually evaluated for proteins relevant to MM and mapped to pathways using Metacore GeneGO. Because both melphalan and doxorubicin produce DNA damage, common processes include DNA damage repair, cell cycle regulation, and apoptosis. Each drug also produces unique phenotypes. For example, doxorubicin resistance is mediated in large part by multidrug resistance proteins; melphalan resistance is marked by increases in redox proteins. LC-MRM of heat shock proteins and Bcl-2 family apoptosis regulators supplements existing data. LC-MRM is also used to examine the response in the expression of proteins involved in ADR at the onset of treatment in naïve MM cells. Conclusions: Proteomics can further elucidate the biological processes involved with ADR in MM to generate candidate biomarkers. LC-MRM is used in cell line models to develop a platform for assessing panels of proteins related to ADR, which can ultimately be translated to assist in clinical decisions for tailoring therapy to the individual patient. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4809. doi:1538-7445.AM2012-4809

Evaluation of protein quantification using standard peptides containing single conservative amino acid replacements

Structural analogs are evaluated as peptide internal standards for protein quantification with liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM); specifically, single conservative amino acid replacements (SCAR) are performed to create tagged standards that differ by the addition or subtraction of a single methylene group in one amino acid side chain. Because the performance of stable isotope-labeled standards (SIS) has been shown to be superior to structural analogs, differences in both development and quantitative performance between assays based on SIS and SCAR peptides are explored. To establish an assay using the structural analogs, analysis of endogenous, SCAR and SIS peptides was performed to examine their ion signal, fragmentation patterns and response in LC-MRM. Performance of SCAR and SIS peptides was compared for quantification of epidermal growth factor receptor from lung cancer cell lysates and immunoglobulin M in the serum of multiple myeloma patients.

Evaluation of Direct Infusion-Multiple Reaction Monitoring Mass Spectrometry for Quantification of Heat Shock Proteins

Protein quantification with liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has emerged as a powerful platform for assessing panels of biomarkers. In this study, direct infusion, using automated, chip-based nanoelectrospray ionization, coupled with MRM (DI-MRM) is used for protein quantification. Removal of the LC separation step increases the importance of evaluating the ratios between the transitions. Therefore, the effects of solvent composition, analyte concentration, spray voltage, and quadrupole resolution settings on fragmentation patterns have been studied using peptide and protein standards. After DI-MRM quantification was evaluated for standards, quantitative assays for the expression of heat shock proteins (HSPs) were translated from LC-MRM to DI-MRM for implementation in cell line models of multiple myeloma. Requirements for DI-MRM assay development are described. Then, the two methods are compared; criteria for effective DI-MRM analysis are reported on the basis of the analysis of HSP expression in digests of whole cell lysates. The increased throughput of DI-MRM analysis is useful for rapid analysis of large batches of similar samples, such as time course measurements of cellular responses to therapy.

Bioanalysis of Biotherapeutics at A Crossroad

Bioanalysis of Biotherapeutics at A Crossroad

Methods for Peptide and Protein Quantitation by Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry

Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633-641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20-30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications.

Laboratory and field evidence of the photonitration of 4-chlorophenol to 2-nitro-4-chlorophenol and of the associated bicarbonate effect

Photochemical processes can decontaminate the aqueous environment from xenobiotics, but they also produce secondary pollutants. This paper presents field and laboratory evidence of the transformation of 4-chlorophenol (4CP) into 2-nitro-4-chlorophenol (2N4CP). Field monitoring of 4CP and 2N4CP was carried out by solid phase extraction coupled with liquid chromatography-multiple reaction monitoring mass spectrometry. Laboratory irradiation experiments were carried out under a UV-Vis lamp, and the time evolution of the compounds of interest was followed by liquid chromatography. The purpose of this study was elucidating the pathways leading to 2N4CP from 4CP in paddy field water. The field monitoring results suggest that 4CP can be transformed into 2N4CP in the paddy field water of the Rhône delta (Southern France). The laboratory study indicates that the transformation can take place via photonitration by (*)NO(2). The nitration process is inhibited by bicarbonate, possibly due to basification that favours the occurrence of the 4-chlorophenolate. The latter could consume (*)NO(2) without being nitrated. Photonitration in the presence of bicarbonate could account for the observed transformation in the field. Photonitration of 4CP to 2N4CP by (*)NO(2) could account for the observed interconversion of the two compounds in paddy fields. The results are of concern because 2N4CP is biorecalcitrant and toxic. Bicarbonate can modulate the photonitration of 4CP into 2N4CP, which can be very significant in bicarbonate-poor waters.

Summary of United States Human Proteome Organisation (HUPO) Symposium Entitled “Standardized Clinical Proteomics Platforms”

Summary of United States Human Proteome Organisation (HUPO) Symposium Entitled “Standardized Clinical Proteomics Platforms”