Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry

Elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues are the biochemical hallmark for patients with X-linked adrenoleukodystrophy (X-ALD). Current methods for the determination of VLCFA levels are laborious and time-consuming. We describe a rapid and easy method using electrospray ionization mass spectrometry (ESI-MS) with deuterated internal standards. VLCFA are hydrolyzed, extracted, and quantified in less than 4h. This includes 2h of hydrolysis and 4min of quantification. We validated the method by analyzing 60 plasma samples from controls and patients with X-ALD or Zellweger syndrome using both the ESI-MS protocol and an established method for VLCFA analysis using gas chromatography (GC). The C26:0 concentrations determined with ESI-MS in plasma and fibroblasts of X-ALD patients are in good agreement with those reported previously for GC and GC-MS. Besides saturated straight chain VLCFA, we also determined the concentrations of the mono-unsaturated VLCFA C24:1 and C26:1 and established that while C24:1 levels are not elevated, C26:1 levels are elevated in both plasma and fibroblasts from X-ALD patients.     

Large-scale determination of SNP allele frequencies in DNA pools using MALDI-TOF mass spectrometry

One of the major challenges in the near future is the identification of genes that contribute to complex disorders. Large scale association studies that utilize a dense map of single nucleotide polymorphisms (SNPs) have been considered as a valuable tool for this purpose. However, genome-wide screens are limited by costs of genotyping thousands of SNPs in a large number of individuals. Here we present a pooling strategy that enables high-throughput SNP validation and determination of allele frequencies in case and control populations. Quantitative analysis of allele frequencies of SNPs in DNA pools is based on matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry of primer extension assays. We demonstrate the accuracy and reliability of this approach on pools of eight previously genotyped individuals with an allele frequency representation in the range of 0.1 to 0.9. The accuracy of measured allele frequencies was shown in DNA pools of 142 to 186 individuals using additional markers. Allele frequencies determined from the pooled samples deviate from the real frequencies by about 3%. The described method reduces costs and time and enables genotyping of up to thousands of samples by taking advantage of the high-throughput MALDI-TOF technology.     

Allele frequencies for 15 short tandem repeat loci in representative sample of Croatian population

Aim

To study the distribution of allele frequencies of 15 short tandem repeat (STR) loci in a representative sample of the Croatian population.

Methods

A total of 195 unrelated Caucasian individuals born in Croatia, from 14 counties and the City of Zagreb, were sampled for the analysis. All the tested individuals were voluntary donors. Buccal swab was used as the DNA source. AmpFlSTR^® Identifiler^® was applied to simultaneously amplify 15 STR loci. Total reaction volume was 12.5 μL. The polymerase chain reaction (PCR) amplification was carried out in PE Gene Amp PCR System Thermal Cycler. Electrophoresis of the amplification products was preformed on an ABI PRISM 3130 Genetic Analyzer. After PCR amplification and separation by electrophoresis, raw data were compiled, analyzed, and numerical allele designations of the profiles were obtained. Deviation from Hardy-Weinberg equilibrium, observed and expected heterozygosity, power of discrimination, and power of exclusion were calculated. Bonferroni’s correction was used before each comparative analysis.

Results

We compared Croatian data with those obtained from geographically neighboring European populations. The significant difference (at P<0.01) in allele frequencies was recorded only between the Croatian and Slovenian populations for vWA locus. There was no significant deviation from Hardy-Weinberg equilibrium for all the observed loci.

Conclusion

Obtained population data concurred with the expected “STR data frame” for this part of Europe.In nearly thirty years since its first formal application (1), DNA analysis has been promoted into a core method for different types of examination, from routine paternity testing and massive identification of human remains to complicated forensic casework analysis (2). Introduction of a set of short tandem repeat (STR) loci as the markers induced a significant progress in this field of science (3-5). DNA typing for forensic, identification, and paternity testing purposes is based on the same techniques that are routinely employed in a wide range of medical and genetic situations, such as diagnosis, population genetic studies, and gene mapping (6). Therefore, importance of understanding the used marker heterogeneity within different populations is constantly emphasized (2,7).Different number and different sets of STR loci in a different number of the individuals of different geographical origin were used in previous studies of the Croatian population. Namely, 8 STR loci have already been employed in a study of the population of Republic of Croatia (8) and 13 CODIS STR loci in the analysis of the population of Southern Croatia (9). Here we have analyzed the distribution of allele frequencies at 15 STR loci in a representative sample of the Croatian population. Additionally, we compared these data with those obtained from geographically neighboring European populations.     

Allele frequencies for 13 STRs loci in a Western Anatolia population and their forensic evaluation

Background: Numerous studies demonstrated that STRs have become powerful tools in forensic case work.

Aim: To profile DNA samples from 104 Turkish males for 13 autosomal, STR markers intended for human identification purposes and to estimate the allele frequency distribution in forensic cases in a Turkish population.

Subjects and methods: Thirteen autosomal STR loci, namely D3S1358, D2S1338, D16S539, D8S1179, D21S11, D18S51, TH01, D13S317, D7S820, CSF1PO, TPOX, D5S818 and FGA, were analysed in a sample of 104 healthy and unrelated Turkish individuals who have been living in the city of ?zmir. All loci were amplified by using AmpFlSTR Identifier Kit. Genetic analysis was carried out on an ABI PRISM 310 Genetic Analyser.

Results: For each locus, 6–15 alleles were found with frequencies ranging from 0.005–0.514 and heterozygosities ranging from 0.686–0.868. The PIC value was highly significant (0.999).

Conclusion: The 13 STR loci in the AmpFlSTR Identifier Kit are suitable for forensic identification and paternity tests due to high heterozygosity. The observed PD value is sufficiently high for human identification purposes. In conclusion, the 13 STR loci seem to be useful markers for personal identification and forensic case work in the Turkish population. The results also demonstrate the importance of region-specific studies.     

成都汉族群体六个短串联重复序列基因频率及其种属特异性研究

目的对成都汉族群体6个短串联重复序列等位基因频率及其种属特异性进行研究,探讨其在法医学中的应用价值。方法用PCR扩增、非变性聚丙烯酰胺凝胶电泳、硝酸银染色技术,对110名成都汉族无血缘关系健康个体的6个短串联重复序列等位基因频率及其种属特异性进行研究。结果D4S2366、D4S2367、D6S474、D6S1281、D2S1396和D20S601在成都汉族群体中等位基因个数分别为7、7、6、7、5、7;观察杂合度分别为0.802、0.708、0.770、0.627、0.542、0.672;个人识别率分别为0.887、0.828、0.849、0.848、0.794、0.865;非父排除率0.602、0.441、0.544、0.325、0.227、0.386。通过种属特异性的评价发现6个短串联重复序列与其他种属间存在较大差异。结论6个短串联重复序列在成都汉族群体中具有较好的遗传多态性及种属特异性,在法医学研究中具有应用价值。     

Discovery of ovarian cancer biomarkers in serum using NanoLC electrospray ionization TOF and FT-ICR mass spectrometry

Treatment of cancer patients is greatly facilitated by detection of the cancer prior to metastasis. One of the obstacles to early cancer detection is the lack of availability of biomarkers with sufficient specificity. With modem differential proteomic techniques, the potential exists to identify high specificity cancer biomarkers. We have delineated a set of protocols for the isolation and identification of serum biomarkers for ovarian cancer that exist in the low molecular weight serum fraction. After isolation of the low molecular weight fraction by ultrafiltration, the potential biomarkers are separated by reversed phase nano liquid chromatography. Detection via TOF or FT-ICR yields a data set for each sample. We compared stage III/IV ovarian cancer serum with postmenopausal age-matched controls. Using bioinformatics tools developed at Mayo, we normalized each sample for intensity and chromatographic alignment. Normalized data sets are subsequently compared and potential biomarkers identified. Several candidate biomarkers were found. One of these contains the sequence of fibrinopeptide-A known to be elevated in many types of cancer including ovarian cancer. The protocols utilized will be examined and would be applicable to a wide variety of cancers or disease states.     

Use of nanoporous alumina surface for desorption electrospray ionization mass spectrometry in proteomic analysis

This paper presents use of a nanoporous alumina surface for desorption electrospray ionization mass spectrometry (DESI MS). The DESI MS performance of the nanoporous alumina surface is compared with that of polymethylmethacrylate (PMMA), polytetrafluroethylene (PTFE) and glass, which are popular surfaces in DESI MS experiments. Optimized operating conditions were determined for each of these surfaces by studying the effects of flow rate, tip to surface and surface to MS capillary distance, and spray angle on the DESI MS performance. The analytes (reserpine and BSA tryptic digest) were analyzed on all the surfaces. The results show that the nanoporous alumina surface offers higher ion intensity and increased peptide detection as compared to the other surfaces. Additionally, comparison of ion intensities obtained from the nanoporus alumina and an alumina film confirms that improved performance is due to the inherent nature of the nanostructured surface. Limits of detection (LODs) were determined for the analytes on all the surfaces. It was observed that the nanoporous alumina surface offers improved limits of detection as compared to other surfaces. Another advantage of the nanoporous alumina surface is that it provides to faster analysis associated with rapid drying of liquid samples on the surface. Additionally, porous alumina surface can be used as a dual ionization platform for combined DESI/LDI analysis for further improved peptide detection in proteomic analysis.     

On-chip coupling of electrochemical pumps and an SU-8 tip for electrospray ionization mass spectrometry

We present the integration of a cyclo olefin copolymer microfluidic chip with electrochemical pumps and an SU-8 tip for electrospray ionization mass spectrometry. The electrochemical pump, using electrolysis as an internal pressure source, was fabricated directly on the surface of the polymer chip. The triangular SU-8 emitter tip was fabricated on a glass wafer using standard photolithography. After release from the glass wafer, this tip was aligned to the microchannel and bonded between two polymer plates. The electrochemical pump and the electrospray tip were tested with electrospray ionization mass spectrometry. Mass spectrometry confirmed the stability of the electrochemical pump and the electrospray tip.     

Allelic loss analysis by denaturing high-performance liquid chromatography and electrospray ionization mass spectrometry

Analysis of allelic imbalance is of great importance for understanding tumorigenesis and the clinical management of malignant disease. Fluorescent-based capillary electrophoresis (CE) of highly polymorphic short tandem repeats (STRs) has become the main method used to detect the loss/gain of alleles. However, there is continued interest in the development of techniques that require no fluorescence and allow the rapid analysis of individual samples. One promising alternative is ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), which is widely available because of its use in denaturing HPLC. Its applicability in combination with ultraviolet (UV) absorbance detection to the efficient separation of di- and tetranucleotide repeats on the short arm of chromosome 11 was tested using 25 matched pairs of normal and ovarian cancer tissues. Loss of heterozygosity (LOH) could be readily identified for all 13 loci tested, based on changes in the ratios between either the alleles or homo- and heteroduplex signals. However, discrimination between noninformative homo- or hemizygous and heterozygous samples was difficult or impossible when HPLC failed to resolve the alleles. Hyphenation of HPLC with electrospray ionization (ESI) quadrupole ion trap (IT) mass spectrometry (MS) not only allowed the identification of coeluting alleles, but also the reliable detection of a 40% reduction of one allele. The size range of DNA fragments amenable to mass spectrometric analysis was effectively tripled to >300 bp by the use of a linear IT and a Taq DNA polymerase cocktail lacking detergents that otherwise adversely affect ESI.     

成都汉族群体五个短串联重复序列基因频率及其种属特异性研究

目的对成都汉族群体5个短串联重复序列（short tandem repeat，STR）基因座的等位基因频率及其种属特异性进行研究，探讨其在法医学中的应用价值。方法用PCR扩增、非变性聚丙烯酰胺凝胶电泳、硝酸银染色方法，对100名成都汉族无血缘关系健康个体的5个STR基因座的等位基因频率及其种属特异性进行研究。结果D18S979、D11S2014、D18S548、D1S1667和GATA164F07在成都汉族群体中等位基因个数分别为6、5、5、7、6；基因型个数分别为12、11、13、19、14；基因型分布符合Hardy-Weinberg平衡定律。通过种属特异性分析发现5个STR基因座中，猴在D18S979、D11S2014和D1S1667基因座均检测出扩增产物，但未在人类基因座分型区内；D18S979位点人类分型区内可检测到牛、狗、鳝鱼有扩增产物，猪、鸭、鼠、兔有微弱扩增产物；D18S548位点人类分型区内只检测到牛有微弱扩增产物；D1S1667位点人类分型区内检测到狗、羊、鳝鱼有扩增产物；GATA164F07位点人类分型区内只检测到狗有微弱扩增产物；泥鳅、鸡、豚鼠在5个基因座均未检测到扩增产物。结论5个短串联重复序列中D18S979、D18S548、D1S1667和GATA164F07在成都汉族群体中具有较好的遗传多态性，D11S2014、D18S548和GATA164F07具有较好的种属特异性，在法医学个人识别和亲子鉴定中有应用价值。     

Analysis of phospholipid species in human blood using normal-phase liquid chromatography coupled with electrospray ionization ion-trap tandem mass spectrometry

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate-acetic acid-ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate-acetic acid-ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile-methanol-ethanol-2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5-2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4-96.5% for retinol (range 100-1000 ng/ml) and 92.7-96.0% for retinyl palmitate (range 5-1000 ng/ml). Inter-assay precision was < or =5.1% and < or =6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.     

Concurrent serotyping and genotyping of pneumococci by use of PCR and electrospray ionization mass spectrometry

A pneumococcal serotyping/genotyping system (PSGS) was developed based upon targeted PCR, followed by electrospray ionization mass spectrometry and amplicon base composition analysis. Eight multiplex PCRs, 32 targeting serotype-determining capsular biosynthetic loci, and 8 targeting multilocus sequence typing (MLST) loci were employed for each of 229 highly diverse Streptococcus pneumoniae isolates. The most powerful aspect of the PSGS system was the identification of capsular serotypes accounting for the majority of invasive and carried pneumococcal strains. Altogether, 45 different serotypes or serogroups were correctly predicted among the 196 resolvable isolates, with only 2 unexpected negative results. All 33 isolates that represented 23 serotypes not included in the PSGS yielded negative serotyping results. A genotyping database was constructed using the base compositions of 65- to 100-bp sections of MLST alleles compiled within http://www.mlst.net. From this database, one or more MLST sequence types (STs) that comprised a PSGS genotype were identified. The end result of more PSGS genotypes (163) than conventional STs actually tested (155) was primarily due to amplification failures of 1 to 3 targets. In many instances, the PSGS genotype could provide resolution of single- and double-locus variants. This molecular serotyping/genotyping scheme is well suited to rapid characterization of large sets of pneumococcal isolates.     

Identification of endosymbionts in ticks by broad-range polymerase chain reaction and electrospray ionization mass spectrometry

Many organisms, such as insects, filarial nematodes, and ticks, contain heritable bacterial endosymbionts that are often closely related to transmissible tickborne pathogens. These intracellular bacteria are sometimes unique to the host species, presumably due to isolation and genetic drift. We used a polymerase chain reaction/electrospray ionization-mass spectrometry assay designed to detect a wide range of vectorborne microorganisms to characterize endosymbiont genetic signatures from Amblyomma americanum (L.), Amblyomma maculatum Koch, Dermacentor andersoni Stiles, Dermacentor occidentalis Marx, Dermacentor variabilis (Say), Ixodes scapularis Say, Ixodes pacificus Cooley & Kohls, Ixodes ricinus (L.), and Rhipicephalus sanguineus (Latreille) ticks collected at various sites and of different stages and both sexes. The assay combines the abilities to simultaneously detect pathogens and closely related endosymbionts and to identify tick species via characterization of their respective unique endosymbionts in a single test.     

Determination of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid in urine using high-performance liquid chromatography and electrospray ionization mass spectrometry.

High-performance liquid chromatography with electrospray ionization mass spectrometry was used to determine 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine. After basic hydrolysis of conjugates, the compound was extracted using SPEC-PLUS-3ML-C18 solid-phase extraction columns. A deuterium labelled internal standard (d3-THC-COOH) was added prior to hydrolysis. Separation was performed on a reversed-phase Zorbax Eclipse XDB-C8 analytical column (150x3.0 mm I.D.) using a gradient program from 60 to 80% acetonitrile (4 mM formic acid) at a flow-rate of 0.5 ml/min. The compounds were detected by single ion monitoring of m/z 345 and m/z 348 for the protonated molecules [THC-COOH+H]+ and [d3-THC-COOH+H]+, respectively. The precision and accuracy were tested on spiked urine samples in the range 2.5-125 ng/ml. The mean recovery was 95% (n = 58), coefficients of variations were 2.2-4.3% and the limit of detection 2 ng/ml. Diagnostic qualifying ions of THC-COOH (m/z 327 and m/z 299) and d3-THC-COOH (m/z 330) were generated using up-front collision-induced dissociation. The relative ion intensities in clinical samples (n = 21) were within +/-20% deviation compared with standards. Using this tolerance and the presence of the ions m/z 327 and m/z 299 at the correct retention times as the acceptance criteria for identification of THC-COOH positive samples, the limit of detection was 15 ng/ml. The LC-MS method complies with the current recommendations on drugs of abuse testing, in which mass spectrometric detection is emphasized.     

Determination of glutathionyl hemoglobin in hemodialysis patients using electrospray ionization liquid chromatography-mass spectrometry.

We first detected glutathionyl hemoglobin (Hb) beta-chain in hemodialysis patients and healthy subjects using electrospray ionization liquid chromatography-mass spectrometry. The ratio of glutathionyl Hb beta-chain to total beta-chain was markedly increased in the hemodialysis patients as compared with healthy subjects. Glutathionyl Hb will be used as a new clinical marker of oxidative stress.     

Determination of ionization efficiency of glycated and non-glycated peptides from the N-terminal of hemoglobin beta-chain by electrospray ionization mass spectrometry

We compared the ionization efficiency of glycated and non-glycated peptides for the HbA1c measurement method developed by Kobold et al. [Clin. Chem., 43 (1997) 1944] based on LC-ESI-MS analysis of the N-terminal peptides of the beta-chains released by cleavage of the hemoglobin with endoproteinase Glu-C. Taking half the peak area of the doubly charged ion and adding it to the area of the singly charged ion, we determined that the slope of the resulting calibration curve was nearly equal to 1, and the reproducibility of the added values was better than the values calculated by the doubly or the singly charged ion alone.     

Proteomic profiling of platelet proteins by trypsin immobilized self-assembled monolayers digestion chip and protein identification using electrospray ionization tandem mass spectrometry

Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition, and other interfacial phenomena. Recently, the bonding of enzyme to SAMs of alkanethiols onto Au electrode surfaces was exploited to produce a bio-sensing system. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling agent. Experimental results have revealed that the X-ray Photoelectron Spectroscopy (XPS) C1s core levels at 286.3 and 286.5 eV (C with N), 288.1 eV (amide bond), and 289.3 eV (carboxyl) illustrate the immobilization of trypsin. These data were also in good agreement with Fourier-Transformed Infrared Reflection-Attenuated Total Reflection (FTIR-ATR) spectra for the peaks valued at 1659.4 cm(-1) (amide I) and 1546.6 cm(-1) (amide II). Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) observations, analytical results have demonstrated the platelet proteins digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, platelet proteins were digested on the trypsin-immobilized SAMs surface, which shows the enzyme digestion ability of the immobilized trypsin. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials.     

Characterization of urinary sulfatides in metachromatic leukodystrophy using electrospray ionization-tandem mass spectrometry

Metachromatic leukodystrophy is an inherited disorder characterized by a deficiency of the lysosomal enzyme arylsulfatase A and the subsequent accumulation of sulfatide in neural and visceral tissues. Clinical diagnosis is usually confirmed by in vitro analysis of arylsulfatase A activity, but may be complicated in cases of arylsulfatase A pseudodeficiency and sphingolipid activator protein deficiency. Large quantities of sulfatide can be detected in the urinary sediment of affected individuals and its measurement can aid in diagnosis. A number of complex methods have been described for the measurement of urinary sulfatide excretion. We have developed a rapid, sensitive, and specific mass spectrometric method for determining urinary sulfatide concentration of metachromatic leukodystrophy patients. Sulfatides are extracted from urine and then directly analyzed using electrospray ionization-tandem mass spectrometry. A sulfatide internal standard has been employed for quantification. The assay has demonstrated significant elevations in the concentrations of several hydroxy and nonhydroxy molecular species of sulfatide in the urine of metachromatic leukodystrophy patients compared to age-matched controls. Analysis of urinary sulfatides in arylsulfatase A pseudodeficiency patients showed a mild elevation in some individuals when related to urinary phosphatidylcholine.     

High-throughput genetic screening using matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a powerful and widespread analytical tool in all fields of life science. Compared with other techniques, its high accuracy and sensitivity makes it a superior method, especially for the analysis of nucleic acids. Recent problems in the analysis of nucleic acids by MALDI-TOF MS can be solved using an automated MALDI-compatible sample-preparation system. Together with the reliable minisequencing assay, high-throughput genotyping of single nucleotide polymorphisms by MALDI-TOF MS is able to become a routine method in research, clinical genetics and diagnostics.