Prerequisites for peptidomic analysis of blood samples: I. Evaluation of blood specimen qualities and determination of technical performance characteristics

Proteomics studies aiming at a detailed analysis of proteins, and peptidomics, aiming at the analysis of the low molecular weight proteome (peptidome) offer a promising approach to discover novel biomarkers valuable for different crucial steps in detection, prevention and treatment of disease. Much emphasis has been given to the analysis of blood, since this source would by far offer the largest number of meaningful biomarker applications. Blood is a complex liquid tissue that comprises cells and extra-cellular fluid. The choice of suitable specimen collection is crucial to minimize artificial occurring processes during specimen collection and preparation (e.g. cell lysis, proteolysis). After specimen collection, sample preparation for peptidomics is carried out by physical methods (filtration, gel-chromatography, precipitation) which allow for separation based on molecular size, with and without immunodepletion of major abundant proteins. Differential Peptide Display (DPD) is an offline-coupled combination of Reversed-Phase-HPLC and MALDI mass spectrometry in combination with in-house developed data display and analysis tools. Identifications of peptides are carried out by additional mass spectrometric methods (e.g. online LC-ESI-MS/MS). In the work presented here, insights into semi-quantitative mass spectrometric profiling of plasma peptides by DPD are given. This includes proper specimen selection (plasma vs. serum), sample preparation, especially peptide extraction, the determination of sensitivity (i.e. by establishing detection limits of exogenously spiked peptides), the reproducibility for individual as well as for all peptides (Coefficient of Variation calculations) and quantification (correlation between signal intensity and concentration). Finally, the implications for clinical peptidomics are discussed.     

Prerequisites for peptidomic analysis of blood samples: II. Analysis of human plasma after oral glucose challenge -- a proof of concept

Mass spectrometric plasma analysis for biomarker discovery has become an exploratory focus in proteomic research: the challenges of analyzing plasma samples by mass spectrometry have become apparent not only since the human proteome organization (HUPO) has put much emphasis on the human plasma proteome. This work demonstrates fundamental proteomic research to reveal sensitivity and quantification capabilities of our Peptidomics technologies by detecting distinct changes in plasma peptide composition in samples after challenging healthy volunteers with orally administered glucose. Differential Peptide Display (DPD) is a technique for peptidomics studies to compare peptides from distinct biological samples. Mass spectrometry (MS) is used as a qualitative and quantitative analysis tool without previous trypsin digestion or labeling of the samples. Circulating peptides (< 15 kDa) were extracted from 1.3 mL plasma samples and the extracts separated by liquid chromatography into 96 fractions. Each fraction was subjected to MALDI MS, and mass spectra of all fractions were combined resulting in a 2D-display of > 2,000 peptides from each sample. Endogenous peptides that responded to oral glucose challenge were detected by DPD of pre-and post-challenge plasma samples from 16 healthy volunteers and subsequently identified by nESI-qTOF MS. Two of the 15 MS peaks that were significantly modulated by glucose challenge were subsequently identified as insulin and C-peptide. These results were validated by using immunoassays for insulin and C-peptide. This paper serves as a proof of principle for proteomic biomarker discovery down to the pM concentration range by using small amounts of human plasma.     

Mass spectrometric detection of siRNA in plasma samples for doping control purposes

Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25–1 nmol mL⁻¹) and performance (precision 11–21%, recovery 23–67%) for typical antisense oligonucleotides currently used in clinical studies.     

A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate pemetrexed in plasma from lung cancer patients

An analytical assay has been developed and validated for ultrafast and high-throughput mass spectrometric determination of pemetrexed concentrations in plasma using matrix assisted laser desorption/ionization–triple quadrupole–tandem mass spectrometry. Patient plasma samples spiked with the internal standard methotrexate were measured by multiple reaction monitoring. The detection limit was 0.4 fmol/μL, lower limit of quantification was 0.9 fmol/μL, and upper limit of quantification was 60 fmol/μL, respectively. Overall observed pemetrexed concentrations in patient samples ranged between 8.7 (1.4) and 142.7 (20.3)?pmol/μL (SD). The newly developed mass spectrometric assay is applicable for (routine) therapeutic drug monitoring of pemetrexed concentrations in plasma from non-small cell lung cancer patients.     

Mass spectrometric imaging of peptide release from neuronal cells within microfluidic devices

Microfluidic devices are well suited for manipulating and measuring mass limited samples. Here we adapt a microfluidic device containing functionalized surfaces to chemically stimulate a small number of neurons (down to a single neuron), collect the release of neuropeptides, and characterize them using mass spectrometry. As only a small fraction of the peptides present in a neuron are released with physiologically relevant stimulations, the amount of material available for measurement is small, thereby requiring minimal sample loss and high-sensitivity detection. Although a number of detection schemes are used with microfluidic devices, mass spectrometric detection is used here because of its high information content, allowing the characterization of the released peptide complement. Rather than using an on-line approach, off-line analysis is used; after collection of the peptides onto a surface, mass spectrometric imaging interrogates that surface to determine the peptides released from the cell. The overall utility of this scheme is demonstrated using several device formats with measurement of neuropeptides released from Aplysia californica bag cell neurons.     

Identification of mammalian cell lines using MALDI-TOF and LC-ESI-MS/MS mass spectrometry

Direct mass spectrometric analysis of complex biological samples is becoming an increasingly useful technique in the field of proteomics. Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) is a rapid and sensitive analytical tool well suited for obtaining molecular weights of peptides and proteins from complex samples. Here, a fast and simple approach to cellular protein profiling is described in which mammalian cells are lysed directly in the MALDI matrix 2,5-dihydroxybenzoic acid (DHB) and mass analyzed using MALDI-time of flight (TOF). Using the unique MALDI mass spectral "fingerprint" generated in these analyses, it is possible to differentiate among several different mammalian cell lines. A number of techniques, including MALDI-post source decay (PSD), MALDI tandem time-of-flight (TOF-TOF), MALDI-Fourier transform ion cyclotron resonance (FTICR), and nanoflow liquid chromatography followed by electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) were employed to attempt to identify the proteins represented in the MALDI spectra. Performing a tryptic digestion of the supernatant of the cells lysed in DHB with subsequent LC-ESI-MS/MS analysis was by far the most successful method to identify proteins.     

Inorganic trace analysis by mass spectrometry

Mass spectrometric methods for the trace analysis of inorganic materials with their ability to provide a very sensitive multielemental analysis have been established for the determination of trace and ultratrace elements in high-purity materials (metals, semiconductors and insulators), in different technical samples (e.g. alloys, pure chemicals, ceramics, thin films, ion-implanted semiconductors), in environmental samples (waters, soils, biological and medical materials) and geological samples. Whereas such techniques as spark source mass spectrometry (SSMS), laser ionization mass spectrometry (LIMS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), glow discharge mass spectrometry (GDMS), secondary ion mass spectrometry (SIMS) and inductively coupled plasma mass spectrometry (ICP-MS) have multielemental capability, other methods such as thermal ionization mass spectrometry (TIMS), accelerator mass spectrometry (AMS) and resonance ionization mass spectrometry (RIMS) have been used for sensitive mono- or oligoelemental ultratrace analysis (and precise determination of isotopic ratios) in solid samples. The limits of detection for chemical elements using these mass spectrometric techniques are in the low ng g⁻¹ concentration range. The quantification of the analytical results of mass spectrometric methods is sometimes difficult due to a lack of matrix-fitted multielement standard reference materials (SRMs) for many solid samples. Therefore, owing to the simple quantification procedure of the aqueous solution, inductively coupled plasma mass spectrometry (ICP-MS) is being increasingly used for the characterization of solid samples after sample dissolution. ICP-MS is often combined with special sample introduction equipment (e.g. flow injection, hydride generation, high performance liquid chromatography (HPLC) or electrothermal vaporization) or an off-line matrix separation and enrichment of trace impurities (especially for characterization of high-purity materials and environmental samples) is used in order to improve the detection limits of trace elements. Furthermore, the determination of chemical elements in the trace and ultratrace concentration range is often difficult and can be disturbed through mass interferences of analyte ions by molecular ions at the same nominal mass. By applying double-focusing sector field mass spectrometry at the required mass resolution—by the mass spectrometric separation of molecular ions from the analyte ions—it is often possible to overcome these interference problems. Commercial instrumental equipment, the capability (detection limits, accuracy, precision) and the analytical application fields of mass spectrometric methods for the determination of trace and ultratrace elements and for surface analysis are discussed.     

Liquid chromatography/tandem mass spectrometric method for the quantification of eight proteolytic fragments of ITIH4 with biomarker potential in human plasma and serum

A liquid chromatography/tandem mass spectrometric (LC/MS/MS) analytical procedure for the quantification of eight proteolytic fragments from inter-alpha-trypsin inhibitor heavy chain 4 (ITIH(4)) in human plasma and serum has been developed. The eight peptide fragments only differ in length at the N-terminus, varying between 21 and 30 amino acid residues. Protein precipitation (PP) with acetonitrile was followed by solid-phase extraction (SPE) on C(2) columns to provide clean extracts. Chromatographic separation of the peptides was performed on a Symmetry 300 C(18) column (50 mm x 2.1 mm i.d., particle size 3.5 microm), using a water/methanol gradient containing 0.25% v/v formic acid. The triple quadrupole mass spectrometer was operated in the positive electrospray ionization (ESI(+)) mode, using multiple reaction monitoring (MRM) for detection. One stable-isotope-labeled analog and two structural analogs were used as internal standards. The method has been completely validated for plasma and partially for serum samples, yielding linear responses in a range up to 100 ng/mL. The lower limit of quantification (LLOQ) in plasma was +/-2 ng/mL for four ITIH(4)-derived peptides and +/-5 ng/mL for the others. The stabilities of the peptides in different environments have been extensively explored. Several peptides showed rapid degradation, especially in the biological matrix at ambient temperature, and preparation on ice was therefore required. The method has been applied for the analysis of several plasma and serum samples from patients with different cancer types. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Rational design of a turn-on fluorescent probe for visualization of GRP78 protein in tumor models

Fluorescence image for accurate tumor label still faces challenges in cancer detection and diagnostics. Emerging evidence is indicating that glucose-regulated protein 78 (GRP78), a stress-inducible protein chaperone, is a great potential biomarker and therapeutic target for cancer. However, currently available probe for image tumor based on GRP78 has not been reported, owning to no obvious strategy in probe design towards this protein. In this paper, a hairpin-shaped peptidyl probe (pepFAM) conjugated with a 5-FAM fluorophore and a dabcyl quencher at both ends was developed, respectively. The probe was designed by performing a traditional fluorescence resonance energy transfer mechanism and employing a GRP78 specifically-binding peptide. Furthermore, the probe was used to specifically image cancer cells, and accurately image xenograft tumors in mice models. The novel fluorescent probe is expected to be a useful tool for the diagnostics of cancer.     

A simple and sensitive assay for the quantitative analysis of paclitaxel in human and mouse plasma and brain tumor tissue using coupled liquid chromatography and tandem mass spectrometry

The development and validation of an assay for the determination of paclitaxel in human plasma, human brain tumor tissue, mouse plasma and mouse brain tumor tissue is described. Paclitaxel was extracted from the matrices using liquid-liquid extraction with tert-butyl methyl ether, followed by chromatographic analysis using an alkaline eluent. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicate that calibration standards in human plasma can be used to quantify paclitaxel in all tested matrices. In human samples, the validated range for paclitaxel was from 0.25-1000 ng ml(-1) using 200 microl plasma aliquots and from 5 to 5000 ng g(-1) using 50 microl tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma). In mice, the ranges were 1-1000 ng ml(-1) and 5-5000 ng g(-1) using 50 microl of mouse plasma and 50 microl of tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma), respectively. The method can be applied to studies generating only small sample volumes (e.g. mouse plasma and tumor tissue), but also to studies in human plasma requiring a lower limit of quantitation. The assay was applied successfully to several studies with both human and mouse samples.     

Highly specific and sensitive liquid chromatography-tandem mass spectrometry method for the determination of 3-desmethylthiocolchicine in human plasma as analyte for the assessment of bioequivalence after oral administration of thiocolchicoside

A sensitive method for the determination of 3-desmethylthiocolchicine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The plasma samples were extracted with ethyl acetate and separated on a Phenomenex Luna C18(2) 5 microm, 150x2 mm column with a mobile phase consisting of acetonitrile-0.005% formic acid (350:650, v/v) at a flow rate of 0.35 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode. TurbolonSpray ionisation was used for ion production. The mean recovery for 3-desmethylthiocolchicine was 70%, with a lower limit of quantification set at 0.39 ng/ml. The increased selectivity of mass spectrometric (MS-MS) detection allowed us to distinguish between thiocolchicoside and its primary metabolite 3-desmethylthiocolchicine in human plasma, thereby giving more insight about the pharmacokinetics of the drug in humans.     

Screening for dihydropyridine calcium channel blockers in plasma by automated solid-phase extraction and liquid chromatography/tandem mass spectrometry

A liquid chromatographic/tandem mass spectrometric (LC/MS/MS) screening method was developed for the screening of 11 calcium channel blockers of the 1,4-dihydropyridine type in plasma samples for forensic and clinical cases. Plasma samples were extracted by automated solid-phase extraction. Analysis was performed using a reversed-phase C(18) column, gradient elution and a triple-quadrupole mass spectrometer with TurboIonSpray source in positive mode and multiple reaction monitoring. This method was found to be selective and sensitive for the detection of the target compounds at their therapeutic plasma concentrations.     

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.     

地球化学样品中钒检测方法的对比

地球化学样品中钒的测定方法主要有原子吸收光谱法、等离子体发射光谱法(ICP-AES)、电感耦合等离子体质谱法(ICP-MS)和X射线荧光光谱法.选用国家一级标样,分别用4种方法测定样品含量值,依次对方法检出限、准确度、精密度、加标回收率作比较.经对比,4种方法测定值与推荐值都基本吻合.等离子体发射光谱法检出限低,线性范围更宽,准确度与精密度更好,更适用于地球化学样品中钒批量的测试.     

Simultaneous detection of intracellular tumor mRNA with bi-color imaging based on a gold nanoparticle/molecular beacon

The successful treatment of most cancers depends on early detection. Tumor mRNA as a specific marker provides new avenues to monitor tumor progression in the early stages and assesses response to treatment. However, single tumor mRNA testing usually yields "false positive" results because cancer is associated with multiple tumor mRNA. It is indispensable to develop simple and effective approaches for the detection of multiple tumor mRNA. In this study, we used a combination of tumor-specific mRNA markers to avoid the inherent limitations associated with the single-marker technique. A gold nanoparticle (AuNP) was assembled with a bi-molecular beacon (bi-MB), and termed AuNP/bi-MB, which simultaneously targeted to two types of tumor mRNA in breast cancer cells. This imaging agent could prevent effectively false positive results and provide comprehensive and dependable information for the early detection of cancer. It would be beneficial to identify the stage of tumor progression and assess treatment decisions with the real-time detection of the relative expression levels of tumor mRNA in cancer cells. This strategy would offer an appealing approach toward the early detection of cancer by using multianalysis of tumor mRNA.     

A high-pressure liquid chromatographic-tandem mass spectrometric method for the determination of ethambutol in human plasma, bronchoalveolar lavage fluid, and alveolar cells

A technique is presented for the specific and sensitive determination of ethambutol concentrations in plasma, bronchoalveolar lavage (BAL), and alveolar cells (AC) using a high-pressure liquid chromatographic (HPLC)-tandem mass spectrometric (MS-MS) method. The preparation of samples requires a deproteinization step with acetonitrile. The retention times for ethambutol, neostigmine bromide, and propranolol are 2.0, 1.4, and 1.1 min, respectively, with a total run time of 2.8 min. The detection limits for ethambutol are 0.05 microg/mL for plasma and 0.005 microg/mL for the BAL supernatants and AC suspensions. The assay has excellent performance characteristics and has been used to support a study of the intrapulmonary pharmacokinetics of ethambutol in human subjects.     

Streamlined in vivo selection and screening of human prostate carcinoma avid phage particles for development of peptide based in vivo tumor imaging agents

Bacteriophage (phage) display has been exploited for the purpose of discovering new cancer specific targeting peptides. However, this approach has resulted in only a small number of tumor targeting peptides useful as in vivo imaging agents. We hypothesize that in vivo screening for tumor uptake of fluorescently tagged phage particles displaying multiple copies of an in vivo selected tumor targeting peptide will expedite the development of peptide based imaging agents. In this study, both in vivo selection and in vivo screening of phage displaying foreign peptides were utilized to best predict peptides with the pharmacokinetic properties necessary for translation into efficacious in vivo imaging agents. An in vivo selection of phage display libraries was performed in SCID mice bearing human PC-3 prostate carcinoma tumors. Eight randomly selected phage clones and four control phage clones were fluorescently labeled with AlexaFluor 680 for subsequent in vivo screening and analyses. The corresponding peptides of six of these phage clones were tested as 111In-labeled peptide conjugates for single photon emission computed tomography (SPECT) imaging of PC-3 prostate carcinomas. Two peptide sequences, G1 and H5, were successful as in vivo imaging agents. The affinities of G1 and H5 peptides for cultured PC-3 cells were then analyzed via cell flow cytometry resulting in Kd values of 1.8 μM and 2.2 μM, respectively. The peptides bound preferentially to prostate tumor cell lines compared to that of other carcinoma and normal cell lines, and H5 appeared to possess cytotoxic properties. This study demonstrates the value of in vivo screening of fluorescently labeled phage for the prediction of the efficacy of the corresponding 111In-labeled synthetic peptide as an in vivo SPECT tumor imaging agent.     

Sample preparation of human serum for the analysis of tumor markers. Comparison of different approaches for albumin and gamma-globulin depletion

LC-MS is a powerful method for the sensitive detection of proteins and peptides in biological fluids. However, the presence of highly abundant proteins often masks those of lower abundance and thus generally prevents their detection and identification in proteomic studies. In human serum the most abundant proteins are albumin and gamma-globulins. We tested several approaches to specifically reduce the level of these proteins based on either specific antibodies, dye ligands (for albumin) and protein A or G (for gamma-globulins). The resulting, depleted serum was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and LC-MS for the residual presence of these abundant proteins as well as for other serum proteins that should remain after depletion. To test the applicability of this method to real-life samples, depleted serum of a cervical cancer patient was analyzed for the presence of a specific tumor marker protein SCCA1 (squamous cell carcinoma antigen 1; P29508), which is present at ng/ml concentrations. The results demonstrate that SCCA1 can be detected by LC-MS in patient serum following depletion of albumin and gamma-globulins thus opening the possibility of screening patient sera for other, so far unknown, tumor markers.     

The further study on radioiodinated peptide Arg-Arg-Leu targeted to neovascularization as well as tumor cells in molecular tumor imaging

The importance of angiogenesis in tumor growth and metastasis has led to develop new imaging tracers to understand angiogenic vasculature. Based on the previous study, we further focused on the tumor molecular imaging application of the novel peptide Arginine-Arginine-Leucine (Tyr-Cys-Gly-Gly-Arg-Arg-Leu-Gly-Gly-Cys, tRRL) in this study. The cytotoxicity of raioiodinated tRRL (¹³¹I-tRRL) in HepG2 cells was assessed by tested cell viability using kit. tRRL was conjugated with fluorescein FITC to observe its binding with tumor cells and human aortic endothelial cells (HAEC) in vitro. Whole body SPECT imaging of varied tumors xenograftes was performed after intravenous injection of ¹³¹I-tRRL for 24 h in BALB/c nude mice. Compared with negative control PBS, small peptide tRRL was of non-cytotoxicity. ¹³¹I-tRRL could lead to significant cytotoxicity on HepG2 cells when the radioactivity was greater than 370 kBq. In vitro binding experiment and cellular uptake results revealed that tRRL could adhere to tumor cells besides tumor derived endothelial cells. In vivo SPECT imaging, ¹³¹I-tRRL mainly accumulated in various tumor tissues, including melanoma, liver cancer and lung cancer bearing mice. In breast cancer xenografte imaging, the tumor has no significant radionuclide accumulation at 24 h after injected of ¹³¹I-tRRL. Radioiodinated tRRL offers a noninvasive nuclear imaging method for functional molecular imaging of tumors, and may be a promising candidate carrier for tumor targeted therapy.