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Background Cancer cells are characterized by a deregulated cell cycle that facilitates abnormal proliferation by allowing cells to by-pass tightly regulated molecular checkpoints such as the G1/S restriction point. To facilitate early diagnosis and the identification of new drug targets, current research efforts focus on studies that could lead to the development of protein panels that collectively can improve the effectiveness of our response to the detection of a life-threatening disease. Methods Estrogen-responsive MCF-7 cells were cultured and arrested by serum deprivation in the G1-stage of the cell cycle, and fractionated into nuclear and cytoplasmic fractions. The protein extracts were trypsinized and analyzed by liquid chromatography - mass spectrometry (MS), and the data were interpreted with the Thermo Electron Bioworks software. Biological characterization of the data, selection of cancer markers, and identification of protein interaction networks was accomplished with a combination of bioinformatics tools provided by GoMiner, DAVID and STRING. Results The objective of this work was to explore via MS proteomic profiling technologies and bioinformatics data mining whether randomly identified cancer markers can be associated with the G1-stage of the cell cycle, i.e., the stage in which cancer cells differ most from normal cells, and whether any functional networks can be identified between these markers and placed in the broader context of cell regulatory pathways. The study enabled the identification of over 2000 proteins and 153 cancer markers, and revealed for the first time that the G1-stage of the cell cycle is not only a rich source of cancer markers, but also a host to an intricate network of functional relationships within the majority of these markers. Three major clusters of interacting proteins emerged: (a) signaling, (b) DNA repair, and (c) oxidative phosphorylation. Conclusions The identification of cancer marker regulatory components that act not alone, but within networks, represents an invaluable resource for elucidating the moxlecular mechanisms that govern the uncontrolled proliferation of cancer cells, as well as for catalyzing the development of protein panels with biomarker and drug target potential, screening tests with improved sensitivity and specificity, and novel cancer therapies aimed at pursuing multiple drug targets. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-710) contains supplementary material, which is available to authorized users.

A microfluidic device that enables on-chip matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) detection for liquid chromatography (LC) separations is described. The device comprises an array of functional elements to carry out LC separations, integrates a novel microchip-MS interface to facilitate the orthogonal transposition of the microfluidic LC channel into an array of reservoirs, and enables sensitive MALDI-MS detection directly from the chip. Essentially, the device provides a snapshot MALDI-MS map of the content of the separation channel present on the chip. The detection of proteins with biomarker potential from MCF10A breast epithelial cell extracts, and detection limits in the low fmol range, are demonstrated. In addition, the design of the novel LC-MALDI-MS chip entices the promotion of a new concept for performing sample separations within the limited time-frame that accompanies the dead-volume of a separation channel.

A new, high-speed data acquisition system was tested for high storage rate time-of-flight mass spectrometry (TOFMS) detection in capillary electrophoresis (CE). For high spectral acquisition rates of 4 kHz, a spectral storage rate of 80 spectra s(-)(1) was achieved. The resulting detection limit was in the low amol range (10-25 amol) for continuous infusion investigations.

Mass spectrometry (MS) was first used as a detection tool for microfluidic devices in the late nineties (Figeys et al., Anal Chem 69:3153–3160, 1997; Ramsey and Ramsey, Anal Chem 69:1174–1178, 1997; Xue et al., Anal Chem 69:426–430, 1997), and since then, significant efforts have been invested in the further development of efficient microfluidic-MS interfaces and the exploration of microfluidic-MS applicability in biomolecular analysis. Microfluidic devices can be coupled to MS through both electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) interfaces. Both conventional, such as capillary electrophoresis (CE)/high performance liquid chromatography (HPLC) liquid sheath, liquid junction and micro/nano-ESI interfaces, as well as novel interfaces based on unique features enabled by microfabrication, have been developed. Current protein identification protocols proceed through a series of steps that are generally time-consuming and labor-intensive. The development of microfluidic devices with MS detection has facilitated protein analysis by minimizing sample consumption and enabling high-throughput, automatic sample manipulations. In this chapter, the applicability of microfluidic chips with MS detection in proteomics research is reviewed. Applications that focus on protein analysis, including sample pretreatment, proteolytic digestion and separation are described. Recent progress on cell culture and lysis, protein quantitation, and analysis of protein posttranslational modifications is discussed.

Protein glycosylation is involved in a broad range of biological processes that regulate protein function and control cell fate. As aberrant glycosylation has been found to be implicated in numerous diseases, the study and large-scale characterization of protein glycosylation is of great interest not only to the biological and biomedical research community, but also to the pharmaceutical and biotechnology industry. Due to the complex chemical structure and differing chemical properties of the protein/peptide and glycan moieties, the analysis and structural characterization of glycoproteins has been proven to be a difficult task. Large-scale endeavors have been further limited by the dynamic outcome of the glycosylation process itself, and, occasionally, by the low abundance of glycoproteins in biological samples. Recent advances in mass spectrometry (MS) instrumentation, and progress in miniaturized technologies for sample handling, enrichment and separation, have resulted in robust and compelling analysis strategies that effectively address the challenges of the glycoproteome. This review summarizes the key steps that are involved in the development of efficient glycoproteomic analysis methods, and the latest innovations that led to successful strategies for the characterization of glycoproteins and their corresponding glycans. As a follow-up to this work, we review innovative capillary and microfluidic-MS workflows for the identification, sequencing, and characterization of glycoconjugates.

The advance of novel technologies that will enable the detection of large sets of biomarker proteins, to greatly improve the sensitivity and specificity of an assay, represents a major objective in biomedical research. To demonstrate the power of mass spectrometry (MS) detection for large-scale biomarker screening in cancer research, a simple, one-step approach for fast biomarker fingerprinting in complex cellular extracts is described. MCF-7 breast cancer cells were used as a model system. Fast proteomic profiling of whole cellular extracts was achieved on a linear trap quadrupole (LTQ) mass spectrometer by one of the following techniques: (a) data-dependent liquid chromatography (LC)-MS/MS of un-labeled cell extracts, (b) data-dependent LC-MS/MS with pulsed Q dissociation (PQD) detection of iTRAQ labeled samples, and (c) multiple reaction monitoring (MRM)-MS of low abundant proteins that could not be detected with data-dependent MS/MS. The data-dependent LC-MS/MS analysis of MCF-7 cells enabled the identification of 796 proteins (p0.001) and the simultaneous detection of 156 previously reported putative cancer biomarkers. PQD detection of iTRAQ labeled cells resulted in the detection of 389 proteins and 64 putative biomarkers. MRM-MS analysis enabled the successful monitoring of a panel of low-abundance proteins in one single experiment, highlighting the utility of this technique for targeted analysis in cancer investigations. These results demonstrate that MS-based technologies relying on a one-step separation protocol have the potential to revolutionize biomarker research and screening applications by enabling fast, sensitive and reliable detection of large panels of putative biomarkers. To further stimulate the exploration of proteins that have been previously reported in the literature to be differentially expressed in a variety of cancers, an extensive list of approximately 1100 candidate biomarkers has been compiled and included in the manuscript.

Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

Reversible protein phosphorylation represents a key regulatory mechanism that triggers essential cellular signaling events. The large-scale characterization of protein phosphorylation in a cell represents, therefore, the objective of many biological studies that aim at elucidating the complex signaling pathways that are involved in the progression and/or regression of a disease. The recent implementation of novel MS detection strategies has significantly advanced the capabilities for interrogating the complex cellular phosphoproteome. Simultaneously, the current advent of miniaturized technologies has clearly demonstrated the superior performance of microfluidic instrumentation for bioanalytical and biological applications that cope with speed, sensitivity and throughput-related demands. This review aims at providing an update on the latest developments regarding the interfacing of microfluidic devices with MS detection for exploring the challenging area of phosphoproteomics.

The development of novel and reliable technologies for the analysis of proteins and their post-translational modifications, in particular, has recently received much attention and interest. The implementation of a fully integrated microfluidic device interfaced with MS detection for the analysis of phosphoproteins is presented in this paper. The microfluidic platform (3''x1.5'') comprises two individual sample processing systems: one for performing direct sample infusion and one for performing microfluidic LC separations. Various MS detection strategies, specific for the study of post-translational modifications, were conducted using alpha-casein as a model protein. Neutral loss ion mapping, data-dependent triple-play and neutral loss analysis, and in situ dephosphorylation followed by LC separation and MS detection were performed. Consistent results in identifying phosphopeptides with conventional and microfluidic instrumentation have been obtained. Unlike with conventional instrumentation, however, the microfluidic device enabled the completion of each analysis from only a few microliters of sample, in approximately 10-15 min, and on a bioanalytical platform that facilitates multiplexing and disposability, and thus high-throughput, contamination-free analysis.

Examination of the electrospray ionization product ion spectra of 1,2-dihydropyridinyl and 4-aryl-1,2-dihydropyridinyl derivatives bearing a 1-cyclopropyl or 1-trans-2-phenylcyclopropyl group has led to the characterization of unexpected fragment ions. For example, the base peak at m/z 156 present in the product ion spectrum of trans-1-(2-phenylcyclopropyl)-4-phenyl-1,2-dihydropyridine proved not to be the expected 4-phenylpyridinium species but rather the isomeric 3-phenyl-5-azoniafulvenyl species. The results of studies with a series of structural and isotopically labeled analogs require a novel fragmentation pathway to account for the formation of this and related fragment ions. One possible pathway is based on an initial 1,5-sigmatropic shift of a cyclopropylmethylene hydrogen atom that is accompanied by opening of the cyclopropyl ring. The resulting eniminium intermediates then fragment to yield the 5-azoniafulvenyl species.

Instrument miniaturization is one way of addressing the issues of sensitivity, speed, throughput, and cost of analysis in DNA diagnostics, proteomics, and related biotechnology areas. Microfluidics is of special interest for handling very small sample amounts, with minimal concerns related to sample loss and cross-contamination, problems typical for standard fluidic manipulations. Furthermore, the small footprint of these microfabricated structures leads to instrument designs suitable for high-density, parallel sample processing, and high-throughput analyses. In addition to miniaturized systems designed with optical or electrochemical detection, microfluidic devices interfaced to mass spectrometry have also been demonstrated. Instruments for automated sample infusion analysis are now commercially available, and microdevices utilizing chromatographic or capillary electrophoresis separation techniques are under development. This review aims at documenting the technologies and applications of microfluidic mass spectrometry for the analysis of proteomic samples.

A comprehensive characterization of expressed proteins in a cell lysate or organism, today represents a high priority goal requiring high-throughput instrumentation and rapid analysis protocols. In this presentation, we report on the development of new microfabricated analytical platforms interfaced to electrospray ionization mass spectrometry (ESI-MS) that facilitate on-line analysis of peptide samples. In particular, liquid phase separation elements, microdigestion units, pumping units and electrospray elements were fabricated on a unique microchip platform, and the over-all performance of the integrated system evaluated.

A novel microchip device for electrospray ionization has been fabricated and interfaced to a time-of-flight mass spectrometer. Fluid is electrokinetically transported through the chip to a fine fused-silica capillary inserted directly into a channel at the edge of the device. Electrospray is established at the tip of the capillary, which assures a stable, efficient spray. The electric potential necessary for electrospray generation and the voltage drop for electroosmotic pumping are supplied through an electrically permeable glass membrane contacting the fluidic channel holding the capillary. The membrane is fabricated on the microchip using standard photolithographic and wet chemical etching techniques. Performance relative to other microchip electrospray sources has been evaluated and the device tested for potential use as a platform for on-line electrophoretic detection. Sensitivity was found to be approximately three orders of magnitude better than spraying from the flat edge of the chip. The effect of the capillary on electroosmotic flow was examined both experimentally and theoretically.

The effect of the electrospray ionization (ESI) needle voltage on the electroosmotic flow (EOF) in capillary electrophoresis (CE)-mass spectrometry (MS) was investigated. The radial electric field that penetrates across the CE capillary wall imposed by the ESI needle voltage modifies the typical EOF. This effect was investigated for buffers commonly used in CE-MS. Variations as high as ±30% were observed.

An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17beta-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of4%. The reproducibility of protein identifications was approximately 60%-67% between duplicate, and approximately 50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate approximately 2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, approximately 16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up- or down-regulated.

Background The discovery of novel protein biomarkers is essential in the clinical setting to enable early disease diagnosis and increase survivability rates. To facilitate differential expression analysis and biomarker discovery, a variety of tandem mass spectrometry (MS/MS)-based protein profiling techniques have been developed. For achieving sensitive detection and accurate quantitation, targeted MS screening approaches, such as multiple reaction monitoring (MRM), have been implemented. Methods MCF-7 breast cancer protein cellular extracts were analyzed by 2D-strong cation exchange (SCX)/reversed phase liquid chromatography (RPLC) separations interfaced to linear ion trap MS detection. MS data were interpreted with the Sequest-based Bioworks software (Thermo Electron). In-house developed Perl-scripts were used to calculate the spectral counts and the representative fragment ions for each peptide. Results In this work, we report on the generation of a library of 9,677 peptides (p < 0.001), representing ~1,572 proteins from human breast cancer cells, that can be used for MRM/MS-based biomarker screening studies. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum (p-value, DeltaM, Xcorr, DeltaCn, Sp, no. of matching a, b, y ions in the spectrum), the retention time, and the top 10 most intense product ions that correspond to a given peptide. Only proteins identified by at least two spectral counts are listed. The experimental distribution of protein frequencies, as a function of molecular weight, closely matched the theoretical distribution of proteins in the human proteome, as provided in the SwissProt database. The amino acid sequence coverage of the identified proteins ranged from 0.04% to 98.3%. The highest-abundance proteins in the cellular extract had a molecular weight (MW) Conclusion Preliminary experiments have demonstrated that putative biomarkers, that are not detectable by conventional data dependent MS acquisition methods in complex un-fractionated samples, can be reliable identified with the information provided in this library. Based on the spectral count, the quality of a tandem mass spectrum and the m/z values for a parent peptide and its most abundant daughter ions, MRM conditions can be selected to enable the detection of target peptides and proteins.