Pumping iron: A multi-omics analysis of two extremophilic algae reveals iron economy management.

Marine algae are responsible for half of the world's primary productivity, but this critical carbon sink is often constrained by insufficient iron. One species of marine algae, Dunaliella tertiolecta, is remarkable for its ability to maintain photosynthesis and thrive in low-iron environments. A related species, Dunaliella salina Bardawil, shares this attribute but is an extremophile found in hypersaline environments. To elucidate how algae manage their iron requirements, we produced high-quality genome assemblies and transcriptomes for both species to serve as a foundation for a comparative multiomics analysis. We identified a host of iron-uptake proteins in both species, including a massive expansion of transferrins and a unique family of siderophore-iron-uptake proteins. Complementing these multiple iron-uptake routes, ferredoxin functions as a large iron reservoir that can be released by induction of flavodoxin. Proteomic analysis revealed reduced investment in the photosynthetic apparatus coupled with remodeling of antenna proteins by dramatic iron-deficiency induction of TIDI1, which is closely related but identifiably distinct from the chlorophyll binding protein, LHCA3. These combinatorial iron scavenging and sparing strategies make Dunaliella unique among photosynthetic organisms.

Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment.

There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity in a cell-type-specific manner to better understand and predict the function of complex biological systems such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverage due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (microdroplet processing in one pot for trace samples), multiplexed isobaric labeling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram levels of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5000 unique proteins from a small-sized human pancreatic tissue pixel (∼60,000 µm2) and differentiate unique protein abundance patterns in pancreas. Furthermore, the use of the microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.

Interlaboratory Comparison of Antibody-Free LC-MS/MS Measurements of C-peptide and Insulin.

BACKGROUND: The enhanced precision and selectivity of liquid chromatography-tandem mass spectrometry (LC-MS/MS) makes it an attractive alternative to certain clinical immunoassays. Easily transferrable work flows could help facilitate harmonization and ensure high-quality patient care. We aimed to evaluate the interlaboratory comparability of antibody-free multiplexed insulin and C-peptide LC-MS/MS measurements. METHODS: The laboratories that comprise the Targeted Mass Spectrometry Assays for Diabetes and Obesity Research (TaMADOR) consortium verified the performance of a validated peptide-based assay (reproducibility, linearity, and lower limit of the measuring interval [LLMI]). An interlaboratory comparison study was then performed using shared calibrators, de-identified leftover laboratory samples, and reference materials. RESULTS: During verification, the measurements were precise (2.7% to 3.7%CV), linear (4 to 15 ng/mL for C-peptide and 2 to 14 ng/mL for insulin), and sensitive (LLMI of 0.04 to 0.10 ng/mL for C-peptide and 0.03 ng/mL for insulin). Median imprecision across the 3 laboratories was 13.4% (inter-quartile range [IQR] 11.6%) for C-peptide and 22.2% (IQR 20.9%) for insulin using individual measurements, and 10.8% (IQR 8.7%) and 15.3% (IQR 14.9%) for C-peptide and insulin, respectively, when replicate measurements were averaged. Method comparison with the University of Missouri reference method for C-peptide demonstrated a robust linear correlation with a slope of 1.044 and r2 = 0.99. CONCLUSIONS: Our results suggest that combined LC-MS/MS measurements of C-peptide and insulin are robust and adaptable and that standardization with a reference measurement procedure could allow accurate and precise measurements across sites, which could be important to diabetes research and help patient care in the future.

Targeted Mass Spectrometry Assays for Specific Quantification of Urinary proPSA Isoforms.

Prostate cancer (PCa) is the second leading cause of male cancer-related deaths in the United States. The pre-mature forms of prostate-specific antigen (PSA), proPSA, were shown to be associated with PCa. However, there is a technical challenge in the development of antibody-based immunoassays for specific recognition of each individual proPSA isoform. Herein, we report the development of highly specific, antibody-free, targeted mass spectrometry assays for simultaneous quantification of [-2], [-4], [-5], and [-7] proPSA isoforms in voided urine. The newly developed proPSA assays capitalize on Lys-C digestion to generate surrogate peptides with appropriate length (9-16 amino acids) along with long-gradient liquid chromatography separation. The assay utility of these isoform markers was evaluated in a cohort of 30 well-established clinical urine samples for distinguishing PCa patients from healthy controls. Under the 95% confidence interval, the combination of [-2] and [-4] proPSA isoforms yields the area under curve (AUC) of 0.86, and the AUC value for the combined all four isoforms was calculated to be 0.85. We have further verified [-2]proPSA, the dominant isoform, in an independent cohort of 34 clinical urine samples. Validation of proPSA isoforms in large-scale cohorts is needed to demonstrate their potential clinical utility.

Rapidly Assessing the Quality of Targeted Proteomics Experiments through Monitoring Stable-Isotope Labeled Standards.

Targeted proteomics experiments based on selected reaction monitoring (SRM) have gained wide adoption in the use of clinical biomarkers, cellular modeling, and numerous other biological experiments due to their highly accurate and reproducible quantification. The quantitative accuracy in targeted proteomics experiments is reliant on the stable-isotope, heavy-labeled peptide standards that are spiked into a sample and used as a reference when calculating the abundance of endogenous peptides. Therefore, the quality of measurement for these standards is a critical factor in determining whether data acquisition was successful. With improved mass spectrometry (MS) instrumentation that enables the monitoring of hundreds of peptides in hundreds to thousands of samples, quality assessment is increasingly important and cannot be performed manually. We present Q4SRM, a software tool that rapidly checks the signal from all heavy-labeled peptides and flags those that fail quality-control metrics. Using four metrics, the tool detects problems with both individual SRM transitions and the collective group of transitions that monitor a single peptide. The program's speed and simplicity enable its use at the point of data acquisition and can be ideally run immediately upon the completion of a liquid chromatography-SRM-MS analysis.

Carrier-Assisted Single-Tube Processing Approach for Targeted Proteomics Analysis of Low Numbers of Mammalian Cells.

Heterogeneity in composition is inherent in all cell populations, even those containing a single cell type. Single-cell proteomics characterization of cell heterogeneity is currently achieved by antibody-based technologies, which are limited by the availability of high-quality antibodies. Herein we report a simple, easily implemented, mass spectrometry (MS)-based targeted proteomics approach, termed cLC-SRM (carrier-assisted liquid chromatography coupled to selected reaction monitoring), for reliable multiplexed quantification of proteins in low numbers of mammalian cells. We combine a new single-tube digestion protocol to process low numbers of cells with minimal loss together with sensitive LC-SRM for protein quantification. This single-tube protocol builds upon trifluoroethanol digestion and further minimizes sample losses by tube pretreatment and the addition of carrier proteins. We also optimized the denaturing temperature and trypsin concentration to significantly improve digestion efficiency. cLC-SRM was demonstrated to have sufficient sensitivity for reproducible detection of most epidermal growth factor receptor (EGFR) pathway proteins expressed at levels ≥30 000 and ≥3000 copies per cell for 10 and 100 mammalian cells, respectively. Thus, cLC-SRM enables reliable quantification of low to moderately abundant proteins in less than 100 cells and could be broadly useful for multiplexed quantification of important proteins in small subpopulations of cells or in size-limited clinical samples. Further improvements of this method could eventually enable targeted single-cell proteomics when combined with either SRM or other emerging ultrasensitive MS detection.

Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples.

Two-dimensional reversed-phase capillary liquid chromatography (2D RPLC) separations have enabled comprehensive proteome profiling of biological systems. However, milligram sample quantities of proteins are typically required due to significant losses during offline fractionation. Such a large sample requirement generally precludes the application samples in the nanogram to low-microgram range. To achieve in-depth proteomic analysis of such small-sized samples, we have developed the nanoFAC (nanoflow Fractionation and Automated Concatenation) 2D RPLC platform, in which the first dimension high-pH fractionation was performed on a 75-µm i.d. capillary column at a 300 nL/min flow rate with automated fraction concatenation, instead of on a typically used 2.1 mm column at a 200 µL/min flow rate with manual concatenation. Each fraction was then fully transferred to the second-dimension low-pH nanoLC separation using an autosampler equipped with a custom-machined syringe. We have found that using a polypropylene 96-well plate as collection device as well as the addition of n-Dodecyl ß-d-maltoside (0.01%) in the collection buffer can significantly improve sample recovery. We have demonstrated the nanoFAC 2D RPLC platform can achieve confident identifications of ∼49,000-94,000 unique peptides, corresponding to ∼6,700-8,300 protein groups using only 100-1000 ng of HeLa tryptic digest (equivalent to ∼500-5,000 cells). Furthermore, by integrating with phosphopeptide enrichment, the nanoFAC 2D RPLC platform can identify ∼20,000 phosphopeptides from 100 µg of MCF-7 cell lysate.

Targeted brain proteomics uncover multiple pathways to Alzheimer's dementia.

OBJECTIVE: Previous gene expression analysis identified a network of coexpressed genes that is associated with ß-amyloid neuropathology and cognitive decline in older adults. The current work targeted influential genes in this network with quantitative proteomics to identify potential novel therapeutic targets. METHODS: Data came from 834 community-based older persons who were followed annually, died, and underwent brain autopsy. Uniform structured postmortem evaluations assessed the burden of ß-amyloid and other common age-related neuropathologies. Selected reaction monitoring quantified cortical protein abundance of 12 genes prioritized from a molecular network of aging human brain that is implicated in Alzheimer's dementia. Regression and linear mixed models examined the protein associations with ß-amyloid load and other neuropathological indices as well as cognitive decline over multiple years preceding death. RESULTS: Average age at death was 88.6 years. Overall, 349 participants (41.9%) had Alzheimer's dementia at death. A higher level of PLXNB1 abundance was associated with more ß-amyloid load (p = 1.0 × 10-7 ) and higher PHFtau tangle density (p = 2.3 × 10-7 ), and the association of PLXNB1 with cognitive decline is mediated by these known Alzheimer's disease pathologies. On the other hand, higher IGFBP5, HSPB2, and AK4 and lower ITPK1 levels were associated with faster cognitive decline, and, unlike PLXNB1, these associations were not fully explained by common neuropathological indices, suggesting novel mechanisms leading to cognitive decline. INTERPRETATION: Using targeted proteomics, this work identified cortical proteins involved in Alzheimer's dementia and begins to dissect two different molecular pathways: one affecting ß-amyloid deposition and another affecting resilience without a known pathological footprint. Ann Neurol 2018;83:78-88.

Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS.

Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nanoflow comprehensive two-dimensional liquid chromatography (nLC×LC) platform top-down mass spectrometry analysis of histone proteoforms. The described two-dimensional LC system combines weak cation exchange chromatography under hydrophilic interaction LC conditions (i.e., charge- and hydrophilicity-based separation) with reversed phase liquid chromatography (i.e., hydrophobicity-based separation). The two independent chemical selectivities were run at nanoflows (300 nL/min) and coupled online with high-resolution mass spectrometry employing ultraviolet photodissociation (UVPD-HRMS). The nLC×LC workflow increased the number of intact protein masses observable relative to one-dimensional approaches and allowed characterization of hundreds of proteoforms starting from limited sample quantities (∼1.5 µg).

A Customizable Flow Injection System for Automated, High Throughput, and Time Sensitive Ion Mobility Spectrometry and Mass Spectrometry Measurements.

To better understand disease conditions and environmental perturbations, multiomic studies combining proteomic, lipidomic, and metabolomic analyses are vastly increasing in popularity. In a multiomic study, a single sample is typically extracted in multiple ways, and various analyses are performed using different instruments, most often based upon mass spectrometry (MS). Thus, one sample becomes many measurements, making high throughput and reproducible evaluations a necessity. One way to address the numerous samples and varying instrumental conditions is to utilize a flow injection analysis (FIA) system for rapid sample injections. While some FIA systems have been created to address these challenges, many have limitations such as costly consumables, low pressure capabilities, limited pressure monitoring, and fixed flow rates. To address these limitations, we created an automated, customizable FIA system capable of operating at a range of flow rates (∼50 nL/min to 500 µL/min) to accommodate both low- and high-flow MS ionization sources. This system also functions at varying analytical throughputs from 24 to 1200 samples per day to enable different MS analysis approaches. Applications ranging from native protein analyses to molecular library construction were performed using the FIA system, and results showed a highly robust and reproducible platform capable of providing consistent performance over many days without carryover, as long as washing buffers specific to each molecular analysis were utilized.

Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway.

Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway as our model. A total of 43 phosphopeptides from the EGFR-MAPK pathway were selected for the study. The recovery and sensitivity of two commonly used enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2), combined with selected reaction monitoring (SRM)-MS were evaluated. The recovery of phosphopeptides by IMAC and TiO2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1 to 100 µg starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 µg peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25 µg range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 10 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.

Residual tissue repositories as a resource for population-based cancer proteomic studies.

BACKGROUND: Mass spectrometry-based proteomics has become a powerful tool for the identification and quantification of proteins from a wide variety of biological specimens. To date, the majority of studies utilizing tissue samples have been carried out on prospectively collected fresh frozen or optimal cutting temperature (OCT) embedded specimens. However, such specimens are often difficult to obtain, in limited in supply, and clinical information and outcomes on patients are inherently delayed as compared to banked samples. Annotated formalin fixed, paraffin embedded (FFPE) tumor tissue specimens are available for research use from a variety of tissue banks, such as from the surveillance, epidemiology and end results (SEER) registries' residual tissue repositories. Given the wealth of outcomes information associated with such samples, the reuse of archived FFPE blocks for deep proteomic characterization with mass spectrometry technologies would provide a valuable resource for population-based cancer studies. Further, due to the widespread availability of FFPE specimens, validation of specimen integrity opens the possibility for thousands of studies that can be conducted worldwide. METHODS: To examine the suitability of the SEER repository tissues for proteomic and phosphoproteomic analysis, we analyzed 60 SEER patient samples, with time in storage ranging from 7 to 32 years; 60 samples with expression proteomics and 18 with phosphoproteomics, using isobaric labeling. Linear modeling and gene set enrichment analysis was used to evaluate the impacts of collection site and storage time. RESULTS: All samples, regardless of age, yielded suitable protein mass after extraction for expression analysis and 18 samples yielded sufficient mass for phosphopeptide analysis. Although peptide, protein, and phosphopeptide identifications were reduced by 50, 20 and 76% respectively, from comparable OCT specimens, we found no statistically significant differences in protein quantitation correlating with collection site or specimen age. GSEA analysis of GO-term level measurements of protein abundance differences between FFPE and OCT embedded specimens suggest that the formalin fixation process may alter representation of protein categories in the resulting dataset. CONCLUSIONS: These studies demonstrate that residual FFPE tissue specimens, of varying age and collection site, are a promising source of protein for proteomic investigations if paired with rigorously verified mass spectrometry workflows.

Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues.

Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low nanograms per milliliter to sub-naograms per milliliter level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundance proteins (e.g., ≤ 100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging, especially for these samples without available antibodies for enrichment. To address this need, we have developed an antibody-independent deep-dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide separation and enrichment combined with precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ∼5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue provides precise quantification of endogenous proteins at the ∼10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibodies are not available.

Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics.

BACKGROUND: Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ~10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for early detection, diagnosis, prognosis or targeted therapy of prostate cancer. Moreover, the SPOP mutation sites are distributed in a relatively short region with multiple lysine residues, posing significant challenges for bottom-up proteomics analysis of the SPOP mutations. METHODS: To address this issue, PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry assays have been developed for quantifying wild-type SPOP protein and 11 prostate cancer-derived SPOP mutations. RESULTS: Despite inherent limitations due to amino acid sequence constraints, all the PRISM-SRM assays developed using Arg-C digestion showed a linear dynamic range of at least two orders of magnitude, with limits of quantification ranged from 0.1 to 1 fmol/µg of total protein in the cell lysate. Applying these SRM assays to analyze HEK293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations; however, at present, it is unknown if this also affects the biological activity of the SPOP protein. CONCLUSIONS: In summary, PRISM-SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, providing significant potential in biomarker development for prostate cancer.

Mining the human urine proteome for monitoring renal transplant injury.

The human urinary proteome provides an assessment of kidney injury with specific biomarkers for different kidney injury phenotypes. In an effort to fully map and decipher changes in the urine proteome and peptidome after kidney transplantation, renal allograft biopsy matched urine samples were collected from 396 kidney transplant recipients. Centralized and blinded histology data from paired graft biopsies was used to classify urine samples into diagnostic categories of acute rejection, chronic allograft nephropathy, BK virus nephritis, and stable graft. A total of 245 urine samples were analyzed by liquid chromatography-mass spectrometry using isobaric Tags for Relative and Absolute Quantitation (iTRAQ) reagents. From a group of over 900 proteins identified in transplant injury, a set of 131 peptides were assessed by selected reaction monitoring for their significance in accurately segregating organ injury causation and pathology in an independent cohort of 151 urine samples. Ultimately, a minimal set of 35 proteins were identified for their ability to segregate the 3 major transplant injury clinical groups, comprising the final panel of 11 urinary peptides for acute rejection (93% area under the curve [AUC]), 12 urinary peptides for chronic allograft nephropathy (99% AUC), and 12 urinary peptides for BK virus nephritis (83% AUC). Thus, urinary proteome discovery and targeted validation can identify urine protein panels for rapid and noninvasive differentiation of different causes of kidney transplant injury, without the requirement of an invasive biopsy.

Capillary Electrophoresis-Nanoelectrospray Ionization-Selected Reaction Monitoring Mass Spectrometry via a True Sheathless Metal-Coated Emitter Interface for Robust and High-Sensitivity Sample Quantification.

A new sheathless transient capillary isotachophoresis (CITP)/capillary zone electrophoresis (CZE)-MS interface, based on a commercially available capillary with an integrated metal-coated ESI emitter, was developed in this study aiming at overcoming the reproducibility and ruggedness problems suffered to a certain degree by almost all the available CE-MS interfaces, and pushing the CE-MS technology suitable for routine sample analysis with high sensitivity. The new CITP/CZE-MS interface allows the electric contact between ESI voltage power supply and the CE separation liquid by using a conductive liquid that comes in contact with the metal-coated surface of the ESI emitter, making it a true sheathless CE-MS interface. Stable electrospray was established by avoiding the formation of gas bubbles from electrochemical reaction inside the CE capillary. Crucial operating parameters, such as sample loading volume, flow rate, and separation voltage, were systematically evaluated for their effects on both CITP/CZE separation efficiency and MS detection sensitivity. Around one hundred CITP/CZE-MS analyses can be easily achieved by using the new sheathless CITP/CZE interface without a noticeable loss of metal coating on the ESI emitter surface, or degrading of the ESI emitter performance. The reproducibility in analyte migration time and quantitative performance of the new interface was experimentally evaluated to demonstrate a LOQ below 5 attomole.

Comprehensive quantitative analysis of ovarian and breast cancer tumor peptidomes.

Aberrant degradation of proteins is associated with many pathological states, including cancers. Mass spectrometric analysis of tumor peptidomes, the intracellular and intercellular products of protein degradation, has the potential to provide biological insights on proteolytic processing in cancer. However, attempts to use the information on these smaller protein degradation products from tumors for biomarker discovery and cancer biology studies have been fairly limited to date, largely due to the lack of effective approaches for robust peptidomics identification and quantification and the prevalence of confounding factors and biases associated with sample handling and processing. Herein, we have developed an effective and robust analytical platform for comprehensive analyses of tissue peptidomes, which is suitable for high-throughput quantitative studies. The reproducibility and coverage of the platform, as well as the suitability of clinical ovarian tumor and patient-derived breast tumor xenograft samples with postexcision delay of up to 60 min before freezing for peptidomics analysis, have been demonstrated. Moreover, our data also show that the peptidomics profiles can effectively separate breast cancer subtypes, reflecting tumor-associated protease activities. Peptidomics complements results obtainable from conventional bottom-up proteomics and provides insights not readily obtainable from such approaches.

Orthogonal Injection Ion Funnel Interface Providing Enhanced Performance for Selected Reaction Monitoring-Triple Quadrupole Mass Spectrometry.

The electrodynamic ion funnel facilitates efficient focusing and transfer of charged particles in the higher-pressure regions (e.g., ion source interfaces) of mass spectrometers, thus providing increased sensitivity. An "off-axis" ion funnel design has been developed to reduce the source contamination and interferences from, e.g. ESI droplet residue and other poorly focused neutral or charged particles with very high mass-to-charge ratios. In this study, a dual ion funnel interface consisting of an orthogonal higher pressure electrodynamic ion funnel (HPIF) and an ion funnel trap combined with a triple quadrupole mass spectrometer was developed and characterized. An orthogonal ion injection inlet and a repeller plate electrode was used to direct ions to an ion funnel HPIF at a pressure of 9-10 Torr. Key factors for the HPIF performance characterized included the effects of RF amplitude, the DC gradient, and operating pressure. Compared to the triple quadrupole standard interface more than 4-fold improvement in the limit of detection for the direct quantitative MS analysis of low abundance peptides was observed. The sensitivity enhancement in liquid chromatography selected reaction monitoring (LC-SRM) analyses of low-abundance peptides spiked into a highly complex mixture was also compared with that obtained using both a commercial S-lens interface and an in-line dual-ion funnel interface.

Sensitive targeted quantification of ERK phosphorylation dynamics and stoichiometry in human cells without affinity enrichment.

Targeted mass spectrometry is a promising technology for site-specific quantification of posttranslational modifications. However, a major constraint is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents for enrichment. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometry using a sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection, and multiplexing (PRISM). PRISM provides effective enrichment of target peptides into a given fraction from complex mixture, followed by selected reaction monitoring quantification. Direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) was demonstrated from as little as 25 µg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided ∼10-fold higher signal intensities, presumably due to the better peptide recovery of PRISM. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of epidermal growth factor at both the peak activation (10 min) and steady state (2 h). The maximal ERK activation was observed with 0.3 and 3 ng/mL doses for 10 min and 2 h time points, respectively. The dose-response profiles of individual phosphorylated isoforms showed that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than distributed model of ERK phosphorylation. The PRISM-SRM quantification of protein phosphorylation illustrates the potential for simultaneous quantification of multiple PTMs.

Analytical platform evaluation for quantification of ERG in prostate cancer using protein and mRNA detection methods.

BACKGROUND: The established methods for detecting prostate cancer (CaP) are based on tests using PSA (blood), PCA3 (urine), and AMACR (tissue) as biomarkers in patient samples. The demonstration of ERG oncoprotein overexpression due to gene fusion in CaP has thus provided ERG as an additional biomarker. Based on this, we hypothesized that ERG protein quantification methods can be of use in the diagnosis of prostate cancer. METHODS: An antibody-free assay for ERG3 protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERG negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment). Enzyme-linked immunosorbent assay (ELISA), western blot, NanoString, and qRT-PCR were also used in the analysis of these samples. RESULTS: Recombinant ERG3 protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine. The sensitivity of the in-house ELISA was similar to the PRISM-SRM assay, with detection of 30 pg of purified recombinant ERG3 protein and 10,000 VCaP cells. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 100 VCaP cells, in comparison to NanoString methodologies which detected ERG from 10,000 cells. CONCLUSIONS: Based on this data, we propose that the detection of both ERG transcriptional products with RNA-based assays, as well as protein products of ERG using PRISM-SRM assays, may be of clinical value in developing diagnostic and prognostic assays for prostate cancer given their sensitivity, specificity, and reproducibility.