Biomarker assay translation from discovery to clinical studies in cancer drug development: quantification of emerging protein biomarkers.

Many candidate biomarkers emerging from genomics and proteomics research have the potential to serve as predictive indexes for guiding the development of safer and more efficacious drugs. Research and development of biomarker discovery, selection, and clinical qualification, however, is still a relatively new field for the pharmaceutical industry. Advances in technology provide a plethora of analytical tools to discover and analyze mechanism-and-disease-specific biomarkers for drug development. In the discovery phase, differential proteomic analysis using mass spectrometry enables the identification of candidate biomarkers that are associated with a specific mechanism relevant to disease progression and affected by drug treatment. Reliable bioanalytical methods are then developed and implemented to select promising biomarkers for further studies in animals and humans. Quantitative analytical methods capable of generating reliable data constitute a solid basis for statistical assessment of the predictive utility of biomarkers. Biomarker method validation is diverse and for purposes that are very different from those of drug bioanalysis or diagnostic use. Besides being flexible, it should sufficiently demonstrate the method's ability to meet the study intent and the attendant regulatory requirements. Several papers have been published outlining specific requirements for successful biomarker method development and validation using a "Fit-for-Purpose" approach. Many of the challenges faced during biomarker discovery as well as during technology and process translation are discussed in this chapter, including preanalytical planning, assay development, and preclinical and clinical validation. Specific references to protein biomarkers for cancer drug development are also discussed.

Large-scale mapping of human protein-protein interactions by mass spectrometry.

Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

Systematic determination of ion score cutoffs based on calculated false positive rates: application for identifying ubiquitinated proteins by tandem mass spectrometry.

We report a simple approach for determining ion score cutoffs that permit the confident identification of ubiquitinated proteins by tandem mass spectrometry (MS/MS). Initial experiments involving the analysis of gel bands containing multi-Ubiquitin chains with quadrupole time-of-flight and quadrupole ion trap mass spectrometers revealed that standard ion score cutoffs used for database searching were not sufficiently stringent. We also found that false positive and false negative rates (FPR and FNR) varied significantly depending on the cutoff scores used and that appropriate cutoffs could only be determined following a systematic evaluation of false positive rates. When standard cutoff scores were used for the analysis of complex mixtures of ubiquitinated proteins, unacceptably high FPR were observed. Finally, we found that FPR for ubiquitinated proteins are affected by the size of the protein database that is searched. These observations may be applicable for the study of other post-translational modifications.

Mapping protein-protein interactions by mass spectrometry.

Mass spectrometry is currently at the forefront of technologies for mapping protein-protein interactions, as it is a highly sensitive technique that enables the rapid identification of proteins from a variety of biological samples. When used in combination with affinity purification and/or chemical cross-linking, whole or targeted protein interaction networks can be elucidated. Several methods have recently been introduced that display increased specificity and a reduced occurrence of false-positives. In the future, information gained from human protein interaction studies could lead to the discovery of novel pathway associations and therapeutic targets.

PCSK4-null sperm display enhanced protein tyrosine phosphorylation and ADAM2 proteolytic processing during in vitro capacitation.

OBJECTIVE: To study the molecular basis for the accelerated capacitation rate in PCSK4-null sperm. DESIGN: Comparative and controlled experimental research study. SETTING: Academic medical institute. ANIMAL(S): Male mice C57BL/6J wild-type or null congenics for the Pcsk4 allele. INTERVENTION(S): Cauda and epididymal sperm were capacitated for varying times. MAIN OUTCOME MEASURE(S): Differences in sperm protein tyrosine phosphorylation and proteolytic processing of sperm-egg ligands ADAM2 and ADAM3. RESULT(S): The PCSK4-null sperm proteins are hyper-tyrosine phosphorylated during capacitation. This hyperphosphorylation is dependent on protein kinase A (PKA), albumin, and calcium. There is also more ADAM2 proteolytic processing from a 46-kDa form of ADAM2 to a 27-kDa form in PCSK4-null sperm than in wild-type sperm. This processing is dependent on cholesterol efflux. CONCLUSION(S): Lack of PCSK4 is associated with quantitative changes in the phosphorylation and proteolysis of sperm proteins during capacitation; therefore, alterations in signal transduction and proteolytic processing during capacitation may underlie the fertilization incompetence of PCSK4-null sperm. More investigation is needed to determine how and to what extent these changes might contribute to the loss of fertilizing ability of PCSK4-null sperm.

Identification of lysines within membrane-anchored Mga2p120 that are targets of Rsp5p ubiquitination and mediate mobilization of tethered Mga2p90.

Mga2p90 is an endoplasmic reticulum (ER)-localized transcription factor that is released from the ER membrane by a unique ubiquitin (Ub)-dependent mechanism. Mga2p90 mobilization requires polyubiquitination of its associating membrane-bound Mga2p120 anchor and subsequent Mga2p120-Mga2p90 complex disassembly that is mediated by ATPase Cdc48p and its heteromeric Ub-binding adaptor Npl4p-Ufd1p. Although previous studies have identified the Ub ligase (i.e., Rsp5p) and ligase-binding site on Mga2p120 that play a role in this process, the amino acids of Mga2p120 that are targets of ubiquitination and promote Mga2p90 mobilization are unknown. We have identified, using mass spectrometry analysis of in vitro ubiquitinated Mga2p120-Mga2p90 complex, that lysine residues 983 and 985 contained within the carboxy-terminal domain of Mga2p120 are Rsp5p-directed Ub-conjugation sites. Mutation of these residues as well as proximally located lysine 980 results in suppression of Mga2p120 ubiquitination in vitro and in vivo, inefficient liberation of Mga2p90 by Cdc48p(Npl4p/Ufd1p)in vitro, and ER retention of Mga2p in cells. Moreover, mga2Delta/spt23ts harboring Rsp5p binding and conjugation mga2 mutants express low OLE1 (an Mga2p90 target gene) transcripts and display reduced growth. We conclude that residues 980, 983, and 985 are targets of Rsp5p-induced polyubiquitination and mediate Cdc48p(Npl4p/Ufd1p)-dependent Mga2p90-Mga2p120 separation and Mga2p90 mobilization.

Glycoproteomic reactor for human plasma.

We describe the development of a glycoproteomic reactor that combines multiple biochemical and chemical protein processing into a single device for the study of N-glycosylated proteins. The glycoproteins are first enriched by concanavalin A affinity chromatography and then transferred onto and efficiently processed in the glycoproteomic reactor. This glycoproteomic reactor combines protein concentration and purification, disulfide bond reduction, peptide-N-glycosidase-mediated (18)O-labeling and deglycosylation, alkylation, tryptic digestion and pH based fractionation in a device that has an interstitial volume (reaction volume) of approximately 1 microL. We demonstrated the potential of the glycoproteomic reactor using human plasma. Under stringent criteria, 82 unique glycopeptides representing 41 unique glycoproteins were identified from as little as 5 microL of human plasma. Our glycoproteomic reactor reduces the sample processing time to less than 1.5 h, reduces the reagent consumption while providing over 1000-fold concentration of the sample, provides efficient removal of high concentration of glycan buffer, and, finally, allows both glycopeptides and nonglycosylated tryptic peptides to be analyzed by the mass spectrometer which provides much greater protein coverage and more reliable identifications.

Differential proteomic screen to evidence proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryos.

Post-translational modification of proteins via ubiquitination plays a crucial role in numerous vital functions of the cell. Polyubiquitination is one of the key regulatory processes involved in regulation of mitotic progression. Here we describe a differential proteomic screen dedicated to identification of novel proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryo. Mutated recombinant His6-tagged ubiquitin (Ubi (K48R)) was added to mitotic extract from which we purified conjugated proteins, as well as associated proteins in nondenaturing conditions by cobalt affinity chromatography. Proteins eluted from Ubi (K48R) supplemented and control extracts were compared by LC-MS/MS analysis after monodimensional SDS-PAGE. A total of 144 proteins potentially ubiquitinated or associated with them were identified. Forty-one percent of these proteins were shown to be involved in ubiquitination and/or proteasomal degradation pathway confirming the specificity of the screen. Twelve proteins, among them ubiquitin itself, were shown to carry a "GG" or "LRGG" remnant tag indicating their direct ubiquitination. Interestingly, sequence analysis of ubiquitinated substrates carrying these tags indicated that in Xenopus cell-free embryo extract supplemented with Ubi (K48R) the majority of polyubiquitination occurred through lysine-11 specific ubiquitin chain polymerization. The potential interest in this atypical form of ubiquitination as well as usefulness of our method in analyzing atypical polyubiquitin species is discussed.

Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry.

Ubiquitination plays an essential role in maintaining cellular homeostasis by regulating a multitude of essential processes. The ability to identify ubiquitinated proteins by MS currently relies on a strategy in which ubiquitinated peptides are identified by a 114.1 Da diglycine (GG) tag on lysine residues, which is derived from the C-terminus of ubiquitin, following trypsin digestion. In the following study, we report a more comprehensive approach for mapping ubiquitination sites by trypsin digestion and MS/MS analysis. We demonstrate that ubiquitination sites can be identified by signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da) on internal lysine residues as well as a GG-tag found on the C-terminus of ubiquitinated peptides. Application of this MS-based approach enabled the identification of 96 ubiquitination sites from proteins purified from human MCF-7 breast cancer cells, representing a 2.4-fold increase in the number of ubiquitination sites that could be identified over standard methods. Our improved MS-based strategy will aid future studies which aim to identify and/or characterize ubiquitinated proteins in human cells.

The proteomic reactor facilitates the analysis of affinity-purified proteins by mass spectrometry: application for identifying ubiquitinated proteins in human cells.

Mass spectrometry (MS) coupled to affinity purification is a powerful approach for identifying protein-protein interactions and for mapping post-translational modifications. Prior to MS analysis, affinity-purified proteins are typically separated by gel electrophoresis, visualized with a protein stain, excised, and subjected to in-gel digestion. An inherent limitation of this series of steps is the loss of protein sample that occurs during gel processing. Although methods employing in-solution digestion have been reported, they generally suffer from poor reaction kinetics. In the present study, we demonstrate an application of a microfluidic processing device, termed the Proteomic Reactor, for enzymatic digestion of affinity-purified proteins for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Use of the Proteomic Reactor enabled the identification of numerous ubiquitinated proteins in a human cell line expressing reduced amounts of the ubiquitin-dependent chaperone, valosin-containing protein (VCP). The Proteomic Reactor is a novel technology that facilitates the analysis of affinity-purified proteins and has the potential to aid future biological studies.

Analysis of protein interaction networks using mass spectrometry compatible techniques.

The ability to map protein-protein interactions has grown tremendously over the last few years, making it possible to envision the mapping of whole or targeted protein interaction networks and to elucidate their temporal dynamics. The use of mass spectrometry for the study of protein complexes has proven to be an invaluable tool due to its ability to unambiguously identify proteins from a variety of biological samples. Furthermore, when affinity purification is combined with mass spectrometry analysis, the identification of multimeric protein complexes is greatly facilitated. Here, we review recent developments for the analysis of protein interaction networks by mass spectrometry and discuss the integration of different bioinformatic tools for predicting, validating, and managing interaction datasets.

Proteomic analysis of ubiquitinated proteins from human MCF-7 breast cancer cells by immunoaffinity purification and mass spectrometry.

Post-translational modification of proteins via the covalent attachment of Ubiquitin (Ub) plays an important role in the regulation of protein stability and function in eukaryotic cells. In the present study, we describe a novel method for identifying ubiquitinated proteins from a complex biological sample, such as a whole cell lysate, using a combination of immunoaffinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We have demonstrated the applicability of this approach by identifying 70 ubiquitinated proteins from the human MCF-7 breast cancer cell line after treatment with the proteasome inhibitor MG132. This method will aid the study of protein ubiquitination and may be used as a tool for the discovery of novel biomarkers that are associated with disease progression.

Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry.

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.

15-Deoxy-delta 12,14-prostaglandins D2 and J2 are potent activators of human eosinophils.

15-Deoxy-Delta(12,14)-PDJ(2) (15d-PGJ(2)) is a degradation product of PGD(2) that has been proposed as an anti-inflammatory compound because of its various inhibitory effects, some of which are mediated by peroxisome proliferator-activated receptor-gamma. In contrast to its reported inhibitory effects on macrophages and other cells, we found that this compound is a potent activator of eosinophils, inducing calcium mobilization, actin polymerization, and CD11b expression. It is selective for eosinophils, having little or no effect on neutrophils or monocytes. 15d-PGJ(2) has an EC(50) of approximately 10 nM, similar to that of its precursor, PGD(2). The concentrations of 15d-PGJ(2) required to activate eosinophils are thus much lower than those required for its anti-inflammatory effects (usually micromolar). 15-Deoxy-Delta(12,14)-prostaglandin D(2) (15d-PGD(2)) is also a potent activator of eosinophils, with an EC(50) about the same as that of PGD(2), whereas Delta(12)-PGJ(2) is slightly less potent. Eosinophils pretreated with PGD(2) no longer respond to 15d-PGJ(2), and vice versa, but in both cases the cells still respond to another eicosanoid proinflammatory mediator, 5-oxo-6,8,11,14-eicosatetraenoic acid. This indicates that the effects of 15d-PGJ(2) are mediated by the DP(2)/chemoattractant receptor-homologous molecule expressed on Th2 cells that has recently been identified in eosinophils. 15d-PGJ(2) is selective for the DP(2) receptor, in that it has no effect on DP(1) receptor-mediated adenylyl cyclase activity in platelets. We conclude that 15d-PGJ(2) and 15d-PGD(2) are selective DP(2) receptor agonists that activate human eosinophils with potencies at least 100 times greater than those for the proposed anti-inflammatory effects of 15d-PGJ(2) on other cells.