Zerumbone acts as a radiosensitizer in head and neck squamous cell carcinoma.

INTRODUCTION: Zerumbone is a phytochemical compound of the ginger plant Zingiber zerumbet with cytotoxic effects in various cancer cell lines. To date, zerumbone has shown an antiproliferative effect in oral squamous cell carcinoma cells lines. However, the effect of combination with radiation or cisplatin in head and neck squamous cell carcinoma (HNSCC) is unclear. The aim of this study was to investigate the effect of zerumbone alone, and in combination with irradiation and cisplatin on HNSCC cell lines. METHODS: The three HNSCC cell lines SCC25, Cal27 and FaDu were treated with zerumbone, radiation and/or cisplatin. Cell viability and clonogenic assays were performed. The interaction between zerumbone and radiation or cisplatin was evaluated using the combination index. Apoptosis was measured by flow cytometry and cell migration was assessed using a wound healing assay. RESULTS: Treatment with zerumbone resulted in a dose dependent induction of cytotoxicity and apoptosis in all three cell lines. The combination with cisplatin revealed a synergistic to additive effect in Cal27. The clonogenic assay showed a significant radiosensitizing effect in all three cell lines. The wound healing assay showed a reduction of cell migration in Cal27. CONCLUSION: The natural compound zerumbone shows a cytotoxic and proapoptotic effect on HNSCC cell lines. Furthermore, zerumbone enhances the radiation effect in all three cell lines and thus may be a suitable candidate for combination therapy in HNSCC.

HS-173, a selective PI3K inhibitor, induces cell death in head and neck squamous cell carcinoma cell lines.

BACKGROUND: The selective PI3K (Phosphatidylinositol 3-kinase) inhibitor HS-173 has anticancer activity in non-small cell lung cancer and pancreatic cancer cells. Of all head and neck squamous cell carcinomas (HNSCC) 20% harbor specific mutations in the genome. The aim of this study was to investigate the effect of HS-173 on HNSCC cell lines. METHODS: The cell lines SCC25, CAL27 and FaDu were incubated with HS-173. Its antiproliferative effect was determined using the CCK­8 cell proliferation assay. Combined incubation with cisplatin was performed and combination index analysis was conducted. To investigate its effect on radiotherapy, cells were irradiated with 2, 4, 6 and 8 Gy, respectively. Synergistic effects of radiation and HS-173 were measured by proliferation assays and clonogenic survival. RESULTS: The use of HS-173 induced significant reduction of cell proliferation across all cell lines. Most interestingly, it showed a synergistic effect with cisplatin treatment. Clonogenic survival revealed a radiosensitizing effect in CAL27 and FaDu cells. The HS-173 caused significant induction of apoptosis in SCC25 and FaDu cells. CONCLUSION: The selective PI3K inhibitor HS-173 is a potent chemosensitizing and also radiosensitizing drug in treatment of HNSCC cell lines and could be an effective treatment in PI3K-mutated HNSCC.

Modification of preoperative radiochemotherapy for esophageal cancer (CROSS protocol) is safe and efficient with no impact on surgical morbidity.

PURPOSE: Neoadjuvant radiochemotherapy (RCTH) is proven to be highly effective in the treatment of esophageal cancer (EC). We investigated oncological outcome and morbidity in patients treated with a modified CROSS protocol followed by esophagectomy at our institution. METHODS: Patients with EC receiving neoadjuvant RCTH with paclitaxel and carboplatin and concurrent radiotherapy (46 Gy) followed by esophagectomy were included in this retrospective analysis. Histopathological response, overall survival (OS) and recurrence-free interval (RFI) as well as perioperative morbidity were investigated. RESULTS: Thirty-six patients (86.1% male, mean age 61.3 years, standard deviation 11.52) received neoadjuvant RCTH before surgery. Sixteen patients (44.4%) were treated for squamous cell cancer, whereas 20 patients (55.6%) had adenocarcinoma. The majority (75%) underwent abdominothoracic esophageal resection. Major complications occurred in 7 patients (19.5%) including anastomotic leakage in 4 patients (11.1%). A R0 resection was achieved in 97.2%. A complete pathological remission was seen in 13 patients (36.1%). Major response, classified as Mandard tumor regression grade 1 and 2, was found in 26 patients (72.2%). Median OS and RFI were not reached. CONCLUSIONS: Neoadjuvant radiotherapy with 46 Gy and concomitant chemotherapy with paclitaxel and carboplatin for the treatment of locally advanced esophageal carcinoma is safe and effective. The results of this modified radiotherapy protocol are encouraging and should be considered in future patient treatment and study designs.

A Diagnostic Algorithm That Combines Quantitative 18F-FDG PET Parameters and Contrast-Enhanced CT Improves Posttherapeutic Locoregional Restaging and Prognostication of Survival in Patients With Esophageal Cancer.

PURPOSE: The aim of this study was to determine whether the combination of contrast-enhanced CT (CE-CT) and quantitative F-FDG PET parameters improves locoregional restaging in esophageal cancer (EC) after neoadjuvant therapy. METHODS: Eighty-eight consecutive patients with locally advanced esophageal cancer, who underwent restaging after neoadjuvant chemotherapy or chemoradiotherapy before esophagectomy, were included in this retrospective study. The diagnostic accuracy of CE-CT, visual F-FDG PET/CT (vPET/CT), and quantitative PET parameters was assessed for T and N staging. Histopathology was used as the reference standard. The prognostic value for recurrence-free survival, cancer-specific survival, and overall survival was assessed using Cox regression analysis. RESULTS: Sensitivity, positive predictive value, and accuracy were 78.8%, 70.2%, and 59.0% (CE-CT), and 81.1%, 81.1%, and 68.2% (vPET/CT) for T staging as well as 59.5%, 75.9%, and 50.0% (CE-CT), and 70.2%, 93.7%, and 67.0% (vPET/CT) for N staging, respectively. Tumor length and metabolic tumor volume (MTV) exhibited an incremental increase with advancing T stages (P = 0.002 and 0.038). Contrast-enhanced CT had the highest sensitivity to differentiate advanced T stages (T3/4 vs 0-2; area under the receiver operating curve [AUC], 0.86; P < 0.001), whereas MTV at a threshold of 5.8 mL had the highest sensitivity to detect complete response (T0 vs 1-4; AUC, 0.77; P = 0.002). Contrast-enhanced CT and MTV combined had an even superior accuracy to predict complete response (AUC, 0.82; P < 0.001). The imaging American Joint Committee on Cancer stage provided a better prognostication of recurrence-free survival, cancer-specific survival, and overall survival than either T stage, N stage derived from CE-CT or vPET/CT, or quantitative PET parameters alone. CONCLUSIONS: Combined CE-CT and MTV had the highest diagnostic accuracy to identify the posttherapeutic T stage, allowing for robust prediction of recurrence and survival.

6-shogaol induces apoptosis and enhances radiosensitivity in head and neck squamous cell carcinoma cell lines.

Ginger (Zingiber officinale Roscoe) is used for a wide array of conditions in traditional medicine in Asia, but little is known about the effect on head and neck cancer. In this study, the effect of two major pharmacologically active compounds of ginger, 6-gingerol and 6-shogaol, were studied on head and neck cancer cell lines. Furthermore, experiments in combination with established treatment methods for head and neck cancer were performed. Proliferation assays showed a dose-dependent reduction of cell viability. Flow cytometry analysis revealed the induction of apoptosis. Western blot analysis indicated that the antiapoptotic protein survivin was suppressed after treatment. Although a combination of 6-shogaol with cisplatin exhibited no synergistic effect, the combination with irradiation showed a synergistic reduction of clonogenic survival. In conclusion, ginger compounds have many noteworthy effects on head and neck cancer cell lines. In particular, the enhancement of radiosensitivity is remarkable.

Inflatable multichannel rectal applicator for adaptive image-guided endoluminal high-dose-rate rectal brachytherapy: design, dosimetric characteristics, and first clinical experiences.

PURPOSE: To investigate the dosimetric results and first clinical experiences with a new designed balloon applicator with adjustable catheters for endoluminal brachytherapy for patients with locally advanced rectal cancer not undergoing surgery. MATERIAL AND METHODS: The applicator consists of an inflatable rectal balloon with six attached Foley catheters used as guidance for the inserted brachytherapy plastic needles. The construction of the applicator and the dosimetric profile in terms of representative dose points in 0, 2, 5, 10 mm ipsilaterally and in 0 mm contralaterally are described. The first clinical outcomes in three patients are reported. RESULTS: For all three patients, a reproducible dose gradient was achieved. The surface dose on the target side was 204 ± 19% of the normalized dose in 5 mm (100%) tissue depth, and 143 ± 8% in 2 mm and 64 ± 3% in 10 mm tissue depth, while the surface dose on the contra-lateral side was 20 ± 8%. After radiochemotherapy with 50 Gy external beam radiotherapy and concomitant administration of capecitabine, a HDR brachytherapy boost in 2-3 fractions of 7-10 Gy each was delivered. All patients achieved a clinical complete response 3 month after the treatment, and no major toxicity was observed. CONCLUSION: The use of the applicator was clinically feasible, and resulted in a stable and reproducible dose distribution. First clinical results are promising.

Effect of thymoquinone on head and neck squamous cell carcinoma cells in vitro: Synergism with radiation.

Thymoquinone (TQ) is the main bioactive constituent present in black seed oil (Nigella sativa); it has shown anti-inflammatory and anti-neoplastic effects in various cancer cell types. The aim of the present study was to investigate the effects of TQ on head and neck squamous cell carcinoma (HNSCC) cell lines, on its own and in combination with radiation and cisplatin, respectively. The SCC25 and CAL27 HNSCC cell lines were treated with TQ alone and in combination with cisplatin or radiation, respectively. Proliferation assays and clonogenic assays were performed. Apoptosis was detected by flow cytometry. TQ exhibited dose-dependent cytotoxicity via apoptosis in the investigated cell lines. In combination with cisplatin, TQ resulted in no significant increase in cytotoxicity. Combined with radiation, TQ significantly reduced clonogenic survival compared with each treatment method alone. TQ is a promising agent in the treatment of head and neck cancer due to its anti-proliferative and radiosensitizing properties. However, the combination of TQ with cisplatin showed no therapeutic benefit in vitro.

Cocaine adulteration.

Cocaine is a naturally occurring and illicitly used psychostimulant drug. Cocaine acts at monoaminergic neurotransmitter transporters to block uptake of the monoamines, dopamine, serotonin and norepinephrine. The resulting increase of monoamines in the extracellular space underlies the positively reinforcing effects that cocaine users seek. In turn, this increase in monoamines underlies the development of addiction, and can also result in a number of severe side effects. Currently, cocaine is one of the most common illicit drugs available on the European market. However, cocaine is increasingly sold in impure forms. This trend is driven by cocaine dealers seeking to increase their profit margin by mixing ("cutting") cocaine with numerous other compounds ("adulterants"). Importantly, these undeclared compounds put cocaine consumers at risk, because consumers are not aware of the additional potential threats to their health. This review describes adulterants that have been identified in cocaine sold on the street market. Their typical pharmacological profile and possible reasons why these compounds can be used as cutting agents will be discussed. Since a subset of these adulterants has been found to exert effects similar to cocaine itself, we will discuss levamisole, the most frequently used cocaine cutting agent today, and its metabolite aminorex.

Role of cancer stem-cell marker doublecortin-like kinase 1 in head and neck squamous cell carcinoma.

BACKGROUND: So far, no data is available on the role of the tumor stem cell marker doublecortin-like kinase 1 (DCLK1) in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to evaluate DCLK1 expression in HNSCC patients that underwent surgery and postoperative radiotherapy, and to assess its potential as a therapeutic target in vitro. METHODS: We immunohistochemically stained for DCLK1 in 127 sections of HNSCC samples obtained during surgery of HNSCC patients and correlated the expression to patients' overall- and disease-free survival, as well as human papilloma virus (HPV) status. Additionally, we compared our survival data with data obtained from The Cancer Genome Atlas (TCGA). The effects of the DCLK1 inhibitor LRRK-2-in-1 on HNSCC cell lines alone and in combination with irradiation. RESULTS: Expression of DCLK1 in 127 patients was associated with poor survival. In particular, DCLK1 expression had a significant impact on survival of oropharyngeal carcinoma patients. Specifically, DCLK1+/HPV- patients had the worst prognosis after simultaneous assessment of DCLK1 and HPV status in comparison to the other three possible DCLK1/HPV constellations. Higher levels of DCLK1 mRNA were also associated with poor clinical outcome. Inhibition of DCLK1 in our HNSCC cell lines led to growth arrest and induction of apoptosis. The combination of DCLK1 inhibition with irradiation had a synergistic effect. CONCLUSION: Firstly, DCLK1 is a prognostic biomarker for shortened survival. Secondly, through inhibition of DCLK1, it may serve as a therapeutic target as well.

Effect of the histone deacetylase inhibitor resminostat on head and neck squamous cell carcinoma cell lines.

BACKGROUND: Carcinogenesis is determined by various epigenetic events, such as histone deacetylation. The purpose of this study was to investigate the effect of the new histone deacetylase inhibitor resminostat on head and neck squamous cell carcinoma (HNSCC) cell lines. METHODS: The cytotoxicity of resminostat and cisplatin on HNSCC cell lines SCC25, CAL27, and FaDu was determined using CCK-8 cell proliferation assay and combination index analysis. Cells were irradiated with 2 to 8 Gray. Apoptosis was measured using flow cytometry and expression of Mcl-1, p-AKT, and survivin was investigated. RESULTS: Treatment with resminostat showed a decrease of cell proliferation of HNSCC cell lines. In addition, a synergistic effect with cisplatin as well as with radiation treatment could be observed. Induction of cell death and dose-dependent downregulation of survivin was evident in all cell lines. CONCLUSION: Resminostat is a promising treatment of HNSCC because of its antiproliferative, chemosensitizing, and radiosensitizing effects. © 2017 Wiley Periodicals, Inc. Head Neck 39: 900-907, 2017.

Application of a Combined Approach to Identify New Psychoactive Street Drugs and Decipher Their Mechanisms at Monoamine Transporters.

Psychoactive compounds can cause acute and long-term health problems and lead to addiction. In addition to well-studied and legally controlled compounds like cocaine, new psychoactive substances (NPS) are appearing in street drug markets as replacement strategies and legal alternatives. NPS are effectively marketed as "designer drugs" or "research chemicals" without any knowledge of their underlying pharmacological mode of action and their potential toxicological effects and obviously devoid of any registration process. As of 2016, the knowledge of structure-activity relationships for most NPS is scarce, and predicting detailed pharmacological activity of newly emerging drugs is a challenging task. Therefore, it is important to combine different approaches and employ biological test systems that are superior to mere chemical analysis in recognizing novel and potentially harmful street drugs. In this chapter, we provide a detailed description of techniques to decipher the molecular mechanism of action of NPS that target the high-affinity transporters for dopamine, norepinephrine, and serotonin. In addition, this chapter provides insights into a combined approach to identify and characterize new psychoactive street drugs of unknown content in a collaboration with the Austrian prevention project "checkit!."

Direct injection of HILIC fractions on the reversed-phase trap column improves protein identification rates for salivary proteins.

Online combination of hydrophilic interaction chromatography (HILIC) and RP chromatography for separation of tryptic peptides is a challenging approach due to the incompatibility of direct loading HILIC fractions on the RP trapping column. High amounts of organic modifiers in loading solvents decrease the binding efficiency of tryptic peptides on C18 phases and lower the number of identifications. A 500 µL loop upfront of the trapping column filled with aqueous mobile phase was employed as a mixing chamber and enabled direct injections and improved saliva protein identification rates of HILIC fractions.

Prognostic value of volumetric PET parameters in unresectable and metastatic esophageal cancer.

PURPOSE: To assess the prognostic value of volumetric parameters measured with PET/CT in patients with advanced or metastatic esophageal cancer (EC). MATERIALS AND METHODS: We identified 71 patients (33 adenocarcinoma [AC] and 38 squamous cell carcinoma [ESCC]) with unresectable or metastatic EC who had PET/CT prior to palliative treatment. Volumetric parameters (metabolic tumor volume [MTV], total lesion glycolysis [TLG], tumor length [TL]) as well as maximum and mean standardized uptake (SUVmax, SUVmean) were obtained from (18)F-FDG PET/CT studies. The correlation between overall survival (OS) and established clinical parameters was assessed using a Cox proportional hazards model. RESULTS: ESCC patients had higher SUVmax and SUVmean compared to AC (p=0.002 and p<0.001, respectively). There was an association of lower SUVmax and SUVmean with metastatic compared to locally advanced tumors (e.g., median SUVmax stage IV: 14.9, 95% confidence interval [95% CI 4.4-35.5] vs. stage IIIA-C: 23.3 [9.2-40.6], p=0.017). TL, MTV and TLG showed an association to OS for all patients and for the subgroup of AC patients (AC; TL: Hazard ratio [HR] 3.23, [95% CI 1.03-10.11], p=0.044; MTV: HR 3.16, [95% CI 1.08-9.23], p=0.035). There was no correlation between PET parameters and survival in ESCC patients. Clinical nodal status was the only clinical variable associated to OS (HR 2.45 [95% CI 1.26-4.75], p=0.008) in AC patients. In a multivariate analysis, nodal status and MTV remained as independent factors associated to OS (N: HR 9.98, [95% CI 1.28-78.11], p=0.028; MTV: HR 1.02, [95% CI 1.01-1.03], p=0.003). CONCLUSIONS: MTV predicted poor OS in patients with advanced AC. No PET parameters were associated to OS in ESCC patients.

Evaluation of Polo-like kinase 1 as a potential therapeutic target in Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare and aggressive malignancy of the skin. Treatment options for MCC include surgery, radiotherapy, and chemotherapy. The purpose of this study was to assess the expression of Polo-like kinase 1 (PLK1) in MCC and the role of the inhibitor, BI2536, as a potential therapeutic option in MCC. METHODS: PLK1 expression was assessed in tissue samples from 28 patients with MCC and 5 healthy skin samples via immunohistochemistry and furthermore in the 2 MCC cell lines, MCC13 and MCC26, via immunoblotting. The impact of increasing doses of BI2536 alone and in combination with cisplatin or irradiation on cell viability was measured using the CCK-8 assay. Colony forming assays were performed to evaluate long-term effects of combination treatments. Additionally, the induction of apoptotic cell death was measured via flow cytometry. RESULTS: PLK1 is moderately to strongly expressed in 75% of the patients with MCC. The PLK1 inhibitor, BI2536, demonstrated marked inhibition of cell proliferation with IC50 in the low nM range (from 10.07-12.39 nM). Furthermore, BI2536 induces apoptosis in MCC cell lines and acts synergistically in combination with irradiation and cisplatin. CONCLUSION: Because of the marked upregulation of PLK1 in MCC tumor samples and potent inhibition of cell proliferation using a specific clinically available inhibitor, targeting of PLK1 qualifies as a potential novel therapeutic strategy in MCC. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1918-E1925, 2016.

Evaluation of spheroid head and neck squamous cell carcinoma cell models in comparison to monolayer cultures.

Two-dimensional (2D) monolayer cell culture models are the most common method used to investigate tumor cells in vitro. In the few last decades, a multicellular spheroid model has gained attention due to its adjacency to tumors in vivo. The aim of the present study was to investigate immunohistochemical differences between these two cell culture systems. The FaDu, CAL27 and SCC25 head and neck squamous cell carcinoma (HNSCC) cell lines were seeded out in monolayer and multicellular spheroids. The FaDu and SCC25 cells were treated with increasing doses of cisplatin and irradiation. CAL27 cells were not used in theproliferation experiments, since the spheroids of CAL27 cells were not able to process the reagent in CCK-8 assays. Furthermore, they were stained to present alterations of the following antigens: Ki-67, vascular endothelial growth factor receptor, epithelial growth factor and survivin. Differences in growth rates and expression patterns were detected in certain HNSCC cell lines. The proliferation rates showed a significant divergence of cells grown in the three-dimensional model compared with cells grown in the 2D model. Overall, multicellular spheroids are a promising method to reproduce the immunohistochemical aspects and characteristics of tumor cells, and may show different response rates to therapeutic options.

Effect of the coffee ingredient cafestol on head and neck squamous cell carcinoma cell lines.

BACKGROUND AND PURPOSE: Cafestol is a diterpene molecule found in coffee beans and has anticarcinogenic properties. The aim of the study was to examine the effects of cafestol in head and neck squamous cell carcinoma (HNSCC) cells. MATERIALS AND METHODS: Three HNSCC cell lines (SCC25, CAL27 and FaDu) were treated with increasing doses of cafestol. Then combination experiments with cisplatin and irradiation were carried out. Drug interactions and possible synergy were calculated using the combination index analysis. Clonogenic assays were performed after irradiation with 2, 4, 6 and 8 Gy, respectively, and the rate of apoptosis was measured with flow cytometry. RESULTS: Treatment of HNSCC cells with cafestol leads to a dose-dependent reduction of cell viability and to induction of apoptosis. Combination with irradiation shows a reduction of clonogenic survival compared to each treatment method alone. In two of the cell lines a significant additive effect was observed. CONCLUSION: Cafestol is a naturally occurring effective compound with growth-inhibiting properties in head and neck cancer cells. Moreover, it leads to a significant inhibition of colony formation.

Absorption kinetics of low-dose chewable aspirin--implications for acute coronary syndromes.

BACKGROUND: This study describes the implications of the pharmacokinetics of low-dose chewable aspirin for acute coronary syndromes. Current guidelines recommend the administration of 162-325 mg aspirin chewing tablets for the treatment of acute myocardial infarction. Although aspirin is widely used and a cornerstone in myocardial infarction, there is no information available on the pharmacokinetics of low doses of chewable aspirin. MATERIALS AND METHODS: This prospective trial assessed the pharmacokinetics of acetylsalicylic acid and its metabolite salicylic acid after intake of 162 mg chewable low-dose aspirin in 35 healthy volunteers. Plasma drug and metabolite levels were analysed using high-performance liquid chromatography, and corresponding pharmacodynamics were determined by impedance aggregometry. RESULTS: Acetylsalicylic acid was rapidly absorbed with a mean Tmax of 27 ± 8 min. Tmax of salicylic acid was 69 ± 21 min. Mean Cmax was 1·8 ± 0·6 mg/L and 7·6 ± 1·4 for acetylsalicylic acid and salicylic acid, respectively. Arachidonic acid-induced aggregation showed maximum platelet inhibition 30 min after drug ingestion. CONCLUSIONS: The characterization of the plasma-time profile fills the gap between the lack of data on pharmacokinetics and the pharmacodynamics and the recommendation for using low-dose chewable aspirin for acute coronary syndromes. We describe for the first time that a 162-mg dose of chewable aspirin is rapidly absorbed and achieves plasma concentrations of the active metabolite salicylic acid required to maximally inhibit platelet aggregation. However, a 162-mg dose is truly a minimum, and doubling this dose might be better for patients with myocardial infarction.

Preoperative treatment with capecitabine, cetuximab and radiotherapy for primary locally advanced rectal cancer--a phase II clinical trial.

BACKGROUND/AIM: To investigate the feasibility and safety of preoperative capecitabine, cetuximab and radiation in patients with MRI-defined locally advanced rectal cancer (LARC, cT3/T4). PATIENTS AND METHODS: 31 patients with LARC were treated with cetuximab and capecitabine concomitantly with 45 Gy radiotherapy and resected by total mesorectal excision. Histopathological response and association with KRAS status was evaluated. RESULTS: R0-resection was possible in 27 of 31 (86%) patients. No complete pathological remission was observed. Radiochemotherapy with capecitabine and cetuximab was safe to administer and diarrhea was the main toxicity. KRAS-status did not correlate to down-staging or pathological response concerning T- or N-stage. CONCLUSION: Neoadjuvant therapy with capecitabine and cetuximab in combination with radiotherapy did not lead to complete pathological remission. Treatment tolerability was excellent and toxicity remained low. KRAS status did not influence treatment outcomes. Capecitabine in combination with radiotherapy remains a standard therapy for locally advanced rectal cancer.

MALDI Orbitrap mass spectrometry for fast and simplified analysis of novel street and designer drugs.

BACKGROUND: New strategies of rapid high-throughput analysis of street drugs without time-consuming sample preparations are necessary due to the massive variety of illicit substances available on the market. METHODS: We used matrix-assisted laser desorption/ionization (MALDI) high-resolution mass spectrometry (HRMS) to identify substances in 74 drug samples obtained from anonymous drug users who participate in the drug prevention initiative "checkit!". We compared our methodology with results derived from "checkit!" where samples are analyzed by high performance liquid chromatography (HPLC) coupled to ultraviolet diode array detection (UV-DAD) as well as single Quad-MS. Reference substances were serially diluted for calibration curves to assess the possibility of obtaining quantitative information with MALDI using an ionic liquid matrix. RESULTS: All drug substances found by "checkit!" analysis were also identified by MALDI HRMS full scan without previous chromatographic separations, including the detection of additionally 16 substances not detected by "checkit!". Reference substances such as cocaine, lysergic acid diethylamide, levamisole and papaverine were detectable using the ionic liquid matrix N,N-diisopropylethylammonium α-cyanohydroxycinnamate. Serial dilutions revealed correlation coefficients ranging from 0.95 to 0.99. CONCLUSION: Considering the growing complexity in the analysis of designer drugs the presented method can be used either in parallel or instead of already established drug identification techniques as a fast and comprehensive primary screening tool.

Empirical multidimensional space for scoring peptide spectrum matches in shotgun proteomics.

Data-dependent tandem mass spectrometry (MS/MS) is one of the main techniques for protein identification in shotgun proteomics. In a typical LC-MS/MS workflow, peptide product ion mass spectra (MS/MS spectra) are compared with those derived theoretically from a protein sequence database. Scoring of these matches results in peptide identifications. A set of peptide identifications is characterized by false discovery rate (FDR), which determines the fraction of false identifications in the set. The total number of peptides targeted for fragmentation is in the range of 10,000 to 20,000 for a several-hour LC-MS/MS run. Typically, <50% of these MS/MS spectra result in peptide-spectrum matches (PSMs). A small fraction of PSMs pass the preset FDR level (commonly 1%) giving a list of identified proteins, yet a large number of correct PSMs corresponding to the peptides originally present in the sample are left behind in the "grey area" below the identity threshold. Following the numerous efforts to recover these correct PSMs, here we investigate the utility of a scoring scheme based on the multiple PSM descriptors available from the experimental data. These descriptors include retention time, deviation between experimental and theoretical mass, number of missed cleavages upon in-solution protein digestion, precursor ion fraction (PIF), PSM count per sequence, potential modifications, median fragment mass error, (13)C isotope mass difference, charge states, and number of PSMs per protein. The proposed scheme utilizes a set of metrics obtained for the corresponding distributions of each of the descriptors. We found that the proposed PSM scoring algorithm differentiates equally or more efficiently between correct and incorrect identifications compared with existing postsearch validation approaches.