Development and validation of a deep eutectic solvent-assisted liquid-liquid extraction method for simultaneous quantification of six steroid hormones in serum by liquid chromatography-tandem mass spectrometry.

Steroid hormones have been reported to be associated with endocrine system diseases. This paper proposes a novel procedure of deep eutectic solvent (DES)-assisted liquid-liquid extraction (LLE) to extract six steroid hormones (including cortisone, cortisol, androstenedione, testosterone, 17-hydroxyprogesterone, and progesterone) from serum coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of five types of L-proline, choline chloride, and citric acid-based DESs were tailored; the DES from L-proline and ethylene glycol at a molar ratio of 1:4 with 20 % acetonitrile was selected as the best-fit assisted solvent for the six steroid hormones compared with other DESs. The parameters for extraction by selected DES were optimized using Box-Behnken design (BBD), and the optimal extraction conditions are 200 µL of acetonitrile, 100 µL of the sample, and 80 µL of DES. Under optimum conditions, the method has good linear calibration ranges (between 0.07 ng mL-1 and 600 ng mL-1), correlation coefficients of determination (r2>0.99), and low limits of quantification (between 0.02 and 0.60 ng mL-1). The extraction recoveries were in the range of 81.84-114.43 %, and the intra-day and inter-day relative standard deviations (RSDs) were less than 10 %.In general, the DES-LC-MS/MS method is a simple and environmentally-friendly method, which can be complementary to the presently available methods for determining steroid hormones in serum.

A new method based on dispersive liquid-liquid microextraction for fat-soluble vitamin determination in serum by LC-MS/MS.

A green and inexpensive pretreatment known as dispersive liquid-liquid microextraction (DLLME) was developed in this assay coupled with the LC-MS/MS method for routine analysis of fat soluble vitamins (FSVs). The technique was performed with methanol as the dispersive solvent and dichloromethane as the extraction solvent. The extraction phase containing FSVs was evaporated to dryness and reconstituted in a mixture of acetonitrile and water. The influence variables concerning the DLLME procedure were optimized. After that, the method was investigated for its applicability in LC-MS/MS analysis. As a result, the parameters were settled for the optimal conditions during the DLLME process. A cheap and lipid-free substance was found as an alternative to serum to eliminate the matrix effect while preparing the calibrators. The method validation indicated that it was suitable for determining FSVs in serum. Moreover, this method was applied successfully to determine serum samples, which was consistent with the literature. In summary, the DLLME method developed in this report was reliable and more cost-effective than the traditional LC-MS/MS method, and could be applied in the future.

Thermomechanical Process Simulation and Experimental Verification for Laser Additive Manufacturing of Inconel®718.

Laser cladding has emerged as a promising technique for custom-built fabrications, remanufacturing, and repair of metallic components. However, frequent melting and solidification in the process cause inevitable residual stresses that often lead to geometric discrepancies and deterioration of the end product. The accurate physical interpretation of the powder consolidation process remains challenging. Thermomechanical process simulation has the potential to comprehend the layer-by-layer additive process and subsequent part-scale implications. Nevertheless, computational accuracy and efficacy have been serious concerns so far; therefore, a hybrid FEM scheme is adopted for efficient prediction of the temperature field, residual stress, and distortion in multilayer powder-fed laser cladding of Inconel®718. A transient material deposition with powder material modeling is schematized to replicate the fabrication process. Moreover, simulation results for residual stress and distortion are verified with in-house experiments, where residual stress is measured with XRD (X-Ray Diffraction) and geometric distortion is evaluated with CMM (Coordinate Measuring Machine). A maximum tensile residual stress of 373 ± 5 MPa is found in the vicinity of the layer right in the middle of the substrate and predicted results are precisely validated with experiments. Similarly, a 0.68 ± 0.01 mm distortion is observed with numerical simulation and showed a precise agreement with experimental data for the same geometry and processing conditions. Conclusively, the implemented hybrid FEM approach demonstrated a robust and accurate prediction of transient temperature field, residual stresses, and geometric distortion in the multilayer laser cladding of Inconel®718.

Synthesis of Icariin-Zinc and its Protective Effect on Exercise Fatigue and Reproductive System Related Glands in Male Rats.

Background: Icariin, a traditional Chinese medicine, plays a protective role in the treatment of exercise fatigue. Zinc, a trace element, plays an important role in the reproductive system. Therefore, we aimed to synthesize an Icariin-Zinc complex (by chemical means) and verify its protective effect on exercise fatigue and the reproductive system using animal experiments. Methods: The icariin-zinc complex was prepared by the reaction of icariin carbonyl and zinc ions (molar ratio 1:3). The molecular formula and structural formula of the complex were identified and tested. Fifty-six rats selected by swimming training were randomly divided into six groups: static control, exercise control, icariin, gluconate zinc (G-Zn group), icariin glucose zinc and icariin-zinc exercise ( low, high dose/L-E group, H-E group) groups. These groups respectively received the following doses: 1 ml/100 g, daily gavage with NS (for the first two groups), 45 mg/kg icariin, 110 mg/kg Gluconate Zinc, Icariin glucose zinc (45 mg/kg Icariin and 110 mg/kg Gluconate Zinc), 60 mg/kg icariin zinc and 180 mg/kg icariin zinc. After 3 weeks of gavage, we conducted 6 weeks of exhaustive swimming training. Test indices such as exhaustive swimming time of rats and body weight were evaluated after the last training exercise. The seminal vesicles, testes, and prostate gland were weighed, and their indices were calculated. The levels of testosterone (in the plasma) and glycogen (in the liver and muscle homogenates) were also evaluated using ELISA. Results: Compared with the static control group, the exhaustive swimming time of the rats in each group was prolonged. Compared with the other groups, the exhaustive swimming time of the L-E and H-E groups was significantly longer (p < 0.01); the Icariin-Zinc complex significantly increased the exhaustive swimming time of the rats. Compared with the static control group, the plasma testosterone content of the L-E and H-E groups increased significantly (p < 0.05). Compared with the exercise control group and G-Zn group, the plasma testosterone content of the H-E group also increased significantly (p < 0.01). The Icariin-Zinc complex significantly increased the serum levels of testosterone in rats. Compared with the control group, the muscle glycogen reserves of each group decreased, indicating that the muscle glycogen reserves of the rats decreased after swimming. Compared with other groups, the Icariin-Zinc complex can reduce the level of glycogen in the muscles, indicating that it can increase the utilization efficiency of glycogen in muscles. Compared with the static control and exercise control groups, the testicular weight of rats in the administration groups increased slightly. The Icariin-Zinc complex increased the testicular weight, indicating that the function of the reproductive system was improved to some extent. Conclusion: Icariin-Zinc can significantly prolong the exhaustive swimming time, improve exercise ability, and increase the plasma testosterone level (which is beneficial for improving the reproductive ability of male rats). Moreover, the beneficial effect of Icariin-Zinc on the glycogen content, testis index, and other reproductive system glands is dose-dependent.

Icariin-conditioned serum engineered with hyaluronic acid promote repair of articular cartilage defects in rabbit knees.

BACKGROUND: Osteochondral defects mostly occur as a result of trauma or articular degeneration. The poor regenerative ability of articular cartilage remains osteochondral defects are a tricky problem to deal with. The modern treatment strategies mainly focus on cartilage tissue engineering with bioactive materials. In this study, we aimed to develop icariin conditioned serum (ICS) together with hyaluronic acid (HA) and determine their ability in reparing osteochondral tissue in a critical-sized defect in rabbit knees. METHODS: Primary chondrocytes were incubated with serum conditioned with icariin at different concentrations, then cell proliferation rates and glycosaminoglycan (GAG) secretion were detected. Rabbits were treated with intra-articular injection of 0.5 mL normal saline (NS), ICS, HA and ICS + HA in the right knee joint, respectively. ICRS scores were used to assess the macroscopic cartilage regeneration. Histological and immunohistochemical analysis including H&E, Safranin O, toluidine blue and collagen II staining were used to determine the repair of cartilage and the regeneration of chondrocytes. RESULTS: Icariin at a low dose of 0.94 g/kg was identified to have significantly promoted the proliferation of chondrocytes and enhance the secretion of GAG. Femoral condyle from rabbits treated by ICS together with HA was observed to be integrated with native cartilage and more subchondral bone regeneration. ICS together with HA could promote repair of the cartilage defect and increase the neoformation of cartilage. CONCLUSIONS: These results demonstrated the potential of ICS combined with HA to promote reparative response in cartilage defects and the possible application in bioactive material based cartilage regeneration therapies.

Detection of Plasma Catecholamines in Human Pheochromocytoma and Primary Hypertension Based on Liquid Chromatography Tandem Mass Spectrometry.

BACKGROUND: The measurement of plasma catecholamines (CAs) including dopamine (DA), epinephrine (E), and norepinephrine (NE) and their derivatives including metanephrine (MN), normetanephrine (NMN), vanillylmandelic acid (VMA), and homovanillic acid (HVA) has been used in the diagnosis of pheochromocytoma and paraganglioma (PPGL) and primary hypertension (PH) but are typically detected individually when clinical testing. In this study, pre-column derivatization with dansyl chloride (DNS-Cl) combined with an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to simultaneously quantify HVA, VMA, MN, NMN, DA, E, and NE in the plasma from patients with PPGL and PH. METHODS: Plasma samples were extracted by acetonitrile and derivatized with DNS-Cl, followed by reverse phase separation and triple quadruple detection. Quantification of the CAs and their derivatives in 10 PPGL, 10 PH, and 100 healthy subjects was performed by UPLC-MS/MS analysis. RESULTS: All the values of detected CAs/derivatives were in the linearity ranges of the fitted curves. The expression levels of the seven CAs in the PPGL and PH patients were significantly higher than the healthy controls, suggesting increased CA production in the former. There were significant differences in plasma NE, NMN, and VMA levels between the PPGL and PH patients, but there was no significant difference in plasma E, MN, DA, and HVA. A discriminant analysis showed that 90% of the final cases were classified correctly based on the detected CAs/derivatives. CONCLUSIONS: Our results show that the combined detection of the seven CAs/derivatives could be used for the clinical diagnosis of PPGL and PH.

Preconditioning in the Rhesus Macaque Induces a Proteomic Signature Following Cerebral Ischemia that Is Associated with Neuroprotection.

Each year, thousands of patients are at risk of cerebral ischemic injury, due to iatrogenic responses to surgical procedures. Prophylactic treatment of these patients as standard care could minimize potential neurological complications. We have shown that protection of brain tissue, in a non-human primate model of cerebral ischemic injury, is possible through pharmacological preconditioning using the immune activator D192935. We postulate that preconditioning with D192935 results in neuroprotective reprogramming that is evident in the brain following experimentally induced cerebral ischemia. We performed quantitative proteomic analysis of cerebral spinal fluid (CSF) collected post-stroke from our previously published efficacy study to determine whether CSF protein profiles correlated with induced protection. Four groups of animals were examined: naïve animals (no treatment or stroke); animals treated with vehicle prior to stroke; D192935 treated and stroked animals, further delineated into two groups, ones that were protected (small infarcts) and those that were not protected (large infarcts). We found that distinct protein clusters defined the protected and non-protected animal groups, with a 16-member cluster of proteins induced exclusively in D192935 protected animals. Seventy percent of the proteins induced in the protected animals have functions that would enhance neuroprotection and tissue repair, including several members associated with M2 macrophages, a macrophage phenotype shown to contribute to neuroprotection and repair during ischemic injury. These studies highlight the translational importance of CSF biomarkers in defining mechanism and monitoring responses to treatment in development of stroke therapeutics.

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.

Targeted proteomic assays for quantitation of proteins identified by proteogenomic analysis of ovarian cancer.

Mass spectrometry (MS) based targeted proteomic methods such as selected reaction monitoring (SRM) are emerging as a promising tool for verification of candidate proteins in biological and biomedical applications. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute has investigated the standardization and analytical validation of the SRM assays and demonstrated robust analytical performance on different instruments across different laboratories. An Assay Portal has also been established by CPTAC to provide the research community a resource consisting of large sets of targeted MS-based assays, and a depository to share assays publicly. Herein, we report the development of 98 SRM assays that have been thoroughly characterized according to the CPTAC Assay Characterization Guidance Document; 37 of these passed all five experimental tests. The assays cover 70 proteins previously identified at the protein level in ovarian tumors. The experiments, methods and results for characterizing these SRM assays for their MS response, repeatability, selectivity, stability, and endogenous detection are described in detail. Data are available via PeptideAtlas, Panorama and the CPTAC Assay Portal.

Dysfunction of PLA2G6 and CYP2C44-associated network signals imminent carcinogenesis from chronic inflammation to hepatocellular carcinoma.

Little is known about how chronic inflammation contributes to the progression of hepatocellular carcinoma (HCC), especially the initiation of cancer. To uncover the critical transition from chronic inflammation to HCC and the molecular mechanisms at a network level, we analyzed the time-series proteomic data of woodchuck hepatitis virus/c-myc mice and age-matched wt-C57BL/6 mice using our dynamical network biomarker (DNB) model. DNB analysis indicated that the 5th month after birth of transgenic mice was the critical period of cancer initiation, just before the critical transition, which is consistent with clinical symptoms. Meanwhile, the DNB-associated network showed a drastic inversion of protein expression and coexpression levels before and after the critical transition. Two members of DNB, PLA2G6 and CYP2C44, along with their associated differentially expressed proteins, were found to induce dysfunction of arachidonic acid metabolism, further activate inflammatory responses through inflammatory mediator regulation of transient receptor potential channels, and finally lead to impairments of liver detoxification and malignant transition to cancer. As a c-Myc target, PLA2G6 positively correlated with c-Myc in expression, showing a trend from decreasing to increasing during carcinogenesis, with the minimal point at the critical transition or tipping point. Such trend of homologous PLA2G6 and c-Myc was also observed during human hepatocarcinogenesis, with the minimal point at high-grade dysplastic nodules (a stage just before the carcinogenesis). Our study implies that PLA2G6 might function as an oncogene like famous c-Myc during hepatocarcinogenesis, while downregulation of PLA2G6 and c-Myc could be a warning signal indicating imminent carcinogenesis.

Integrated Proteogenomic Characterization of Human High-Grade Serous Ovarian Cancer.

To provide a detailed analysis of the molecular components and underlying mechanisms associated with ovarian cancer, we performed a comprehensive mass-spectrometry-based proteomic characterization of 174 ovarian tumors previously analyzed by The Cancer Genome Atlas (TCGA), of which 169 were high-grade serous carcinomas (HGSCs). Integrating our proteomic measurements with the genomic data yielded a number of insights into disease, such as how different copy-number alternations influence the proteome, the proteins associated with chromosomal instability, the sets of signaling pathways that diverse genome rearrangements converge on, and the ones most associated with short overall survival. Specific protein acetylations associated with homologous recombination deficiency suggest a potential means for stratifying patients for therapy. In addition to providing a valuable resource, these findings provide a view of how the somatic genome drives the cancer proteome and associations between protein and post-translational modification levels and clinical outcomes in HGSC. VIDEO ABSTRACT.

Contributions of immunoaffinity chromatography to deep proteome profiling of human biofluids.

Human biofluids, especially blood plasma or serum, hold great potential as the sources of candidate biomarkers for various diseases; however, the enormous dynamic range of protein concentrations in biofluids represents a significant analytical challenge for detecting promising low-abundance proteins. Over the last decade, various immunoaffinity chromatographic methods have been developed and routinely applied for separating low-abundance proteins from the high- and moderate-abundance proteins, thus enabling much more effective detection of low-abundance proteins. Herein, we review the advances of immunoaffinity separation methods and their contributions to the proteomic applications in human biofluids. The limitations and future perspectives of immunoaffinity separation methods are also discussed.

Using the CPTAC Assay Portal to Identify and Implement Highly Characterized Targeted Proteomics Assays.

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and posttranslational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

BACKGROUND: For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT: The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.

An Optimized Informatics Pipeline for Mass Spectrometry-Based Peptidomics.

The comprehensive MS analysis of the peptidome, the intracellular and intercellular products of protein degradation, has the potential to provide novel insights on endogenous proteolytic processing and its utility in disease diagnosis and prognosis. Along with the advances in MS instrumentation and related platforms, a plethora of proteomics data analysis tools have been applied for direct use in peptidomics; however, an evaluation of the currently available informatics pipelines for peptidomics data analysis has yet to be reported. In this study, we began by evaluating the results of several popular MS/MS database search engines, including MS-GF+, SEQUEST, and MS-Align+, for peptidomics data analysis, followed by identification and label-free quantification using the well-established accurate mass and time (AMT) tag and newly developed informed quantification (IQ) approaches, both based on direct LC-MS analysis. Our results demonstrated that MS-GF+ outperformed both SEQUEST and MS-Align+ in identifying peptidome peptides. Using a database established from MS-GF+ peptide identifications, both the AMT tag and IQ approaches provided significantly deeper peptidome coverage and less missing data for each individual data set than the MS/MS methods, while achieving robust label-free quantification. Besides having an excellent correlation with the AMT tag quantification results, IQ also provided slightly higher peptidome coverage. Taken together, we propose an optimized informatics pipeline combining MS-GF+ for initial database searching with IQ (or AMT tag) approaches for identification and label-free quantification for high-throughput, comprehensive, and quantitative peptidomics analysis. Graphical Abstract ᅟ.

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.

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.

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.