Applications of liquid-based separation in conjunction with mass spectrometry to the analysis of forensic evidence.

In the past few years, there has been a significant effort by the forensic science community to develop new scientific techniques for the analysis of forensic evidence. Forensic chemists have been spearheaded to develop information-rich confirmatory technologies and techniques and apply them to a broad array of forensic challenges. The purpose of these confirmatory techniques is to provide alternatives to presumptive techniques that rely on data such as color changes, pattern matching, or retention time alone, which are prone to more false positives. To this end, the application of separation techniques in conjunction with mass spectrometry has played an important role in the analysis of forensic evidence. Moreover, in the past few years the role of liquid separation techniques, such as liquid chromatography and capillary electrophoresis in conjunction with mass spectrometry, has gained significant tractions and have been applied to a wide range of chemicals, from small molecules such as drugs and explosives, to large molecules such as proteins. For example, proteomics and peptidomics have been used for identification of humans, organs, and bodily fluids. A wide range of HPLC techniques including reversed phase, hydrophilic interaction, mixed-mode, supercritical fluid, multidimensional chromatography, and nanoLC, as well as several modes of capillary electrophoresis mass spectrometry, including capillary zone electrophoresis, partial filling, full filling, and micellar electrokenetic chromatography have been applied to the analysis drugs, explosives, and questioned documents. In this article, we review recent (2015-2017) applications of liquid separation in conjunction with mass spectrometry to the analysis of forensic evidence.

Buyid Silk and the Tale of Bibi Shahrbanu: Identification of Biomarkers of Artificial Aging (Forgery) of Silk.

Buyid silk forgery is one of the most famous silk forgeries in the world. In 1924-1925, excavation of the Bibi Shahrbanu site in Iran unearthed several silk textiles. The silks were thought to be of the Buyid period (934-1062 BCE) of the Persian Empire and have since been known as the "Buyid silks". In the 1930s, more silk appeared and was reported as being from the Buyid period as well. Controversy over the authenticity of these silks escalated after the purchase of the silks by museums throughout the world. Extensive investigations of several of these silks have been conducted over the years with respect to iconography, weaving patterns, dyes/mordant, style, and even radiocarbon dating. It was found that most of the silks are not from Buyid period. To test the authenticity of these silk fabrics, the recently developed silk dating technique using amino acid racemization (AAR) in conjunction with capillary electrophoresis mass spectrometry was applied to 13 Buyid silk specimens from the Textile Museum collections. Among these silk specimens, the AAR ratios of only one specimen were consistent with authentic silk fabrics collected from various museums. In addition, the aspartic acid racemization ratio of this specimen was also consistent with its 14C dating. The other "Buyid silks" showed excessive levels of amino acid racemization not only for aspartic acid, but also for phenylalanine and tyrosine, inconsistent with racemization rates of these amino acids in authentic historical silk fabrics. Treatment of modern silk with a base at different pH and temperature reproduced the AAR pattern of the Buyid silks, implying that chemical treatment with a base at relatively high temperatures was perhaps the method used to artificially age these fabrics. The results imply that the racemization ratios of aspartic acid, phenylalanine, and tyrosine can be used as biomarkers for identification of naturally versus artificially aged silk.

MEKC-UV as an effective tool for the separation and identification of explosives, high explosives, and their degradation products in environmental samples.

MEKC has been used in conjunction with UV detection for identification and quantitation of high explosives in environmental samples. To ensure the compatibility of the technique with ESI-MS, perfluorooctanoic acid (PFOA), a volatile micelle, was used. Separation of EPA Method 8330 Mixes A and B using various concentrations of the micelle showed that the 80 mM solution of PFOA was the optimum concentration for the separation of the explosives. MEKC analysis of explosives with ESI-MS under optimum micelle concentration provided excellent results indicating the compatibility of the method with ESI-MS. Finally, the MEKC-UV method was applied to the detection and quantitation of explosives in various environmental samples including water, sand, and soil. The results demonstrate that the MEKC method described herein is a viable technique for detection of explosives in environmental samples using UV detection, while maintaining the compatibility of the technique with MS detection without any modification to the separation method, if laboratories decided to pursue this route in the future.

Ultrafast capillary electrophoresis/mass spectrometry of controlled substances with optical isomer separation in about a minute.

RATIONALE: Analysis of forensic evidence by information-rich technologies such as mass spectrometry (MS) is one of the fastest growing areas in forensic analysis. To provide more accurate identification of forensic evidence, in the past few years there has been a growing interest in moving this technology to the field for on-site, real-time analysis. To this end, several portable mass spectrometers have been introduced; however, the analysis of controlled substances could be complicated by the existence of various isomers including optical isomers in which sentencing may depend on the identification of the isomer. To date very few portable separation devices are capable of separating and identifying the optical isomers. METHODS: In this study, the application of the portable ultrafast capillary electrophoresis (UFCE) to the separation of controlled substances is presented and the results are compared with the results obtained from a bench-top CE system. Both a nominal mass ion trap mass spectrometer and an accurate mass orbitrap mass spectrometer were interfaced with CE using a porous tip capillary. RESULTS: A mixture of several controlled substances can be separated and detected using UFCE/MS in about a minute using field strengths of ≥1000 V/cm. Furthermore, separation and detection of underivatized optical isomers of amphetamine, cathinone, nor-mephedrone, and pregabalin using UFCE/MS can be achieved with an analysis time of less than two minutes. Resolutions of 1.3, 3.7 and 3.8 were achieved for pregabalin, cathinone and nor-mephedrone, respectively, under UFCE/MS conditions. CONCLUSIONS: Amphetamine, cathinone, nor-mephedrone and pregabalin were separated and detected in about a minute, demonstrating the utility of the portable CE instrument for the analysis of controlled substances and their optical isomers. Copyright © 2016 John Wiley & Sons, Ltd.

Compatibility of highly sulfated cyclodextrin with electrospray ionization at low nanoliter/minute flow rates and its application to capillary electrophoresis/electrospray ionization mass spectrometric analysis of cathinone derivatives and their optical isomers.

RATIONALE: Sodium salts of cyclodextrins are commonly used in capillary electrophoresis/mass spectrometry (CE/MS) analysis of illicit drugs and their optical isomers. To avoid the suppression effect of cyclodextrins under electrospray ionization (ESI), the partial filling technique (PFT) is commonly utilized, which has a limited resolution. Low-flow nano-ESI has been shown to reduce the suppression effect of the salts. To test the compatibility of low-flow ESI with a background electrolyte (BGE) containing sodium salts of cyclodextrin, sheathless narrow capillary CE/MS with flow rates of low nanoliters/minute (nL/min) was applied to the separation and detection of cathinones and their positional and optical isomers for the first time. METHODS: Low-flow sheathless CE/MS using a 20-µm-i.d. capillary in conjunction with a porous tip interface was used for the separation of cathinone derivatives and their optical isomers. Highly sulfated γ-cyclodextrin (HS-γ-CD) in conjunction with (+)-18-crown-6-tetracarboxylic acid ((+)-18-C-6-TCA) was used as the BGE and an ion trap mass spectrometer operating in full scan mode was utilized. RESULTS: Utilizing low flow rate (~10 nL/min) sheathless CE/MS, the use of the sodium salt of HS-γ-CD as the BGE was compared with the same solution using PFT. The relative and absolute sensitivity of detection of cathinones were about the same, indicating that under low-flow sheathless CE/MS there was no significant suppression due to the existence of HS-γ-CD in the electrospray process. However, enhanced resolution of cathinone derivatives and their positional and optical isomers was observed when the solution of HS-γ-CD was used as the BGE. The enhanced resolution was because of the presence of the HS-γ-CD in the entire capillary during the analysis. The addition of 15 mM (+)-18-C-6-TCA to the BGE containing HS-γ-CD further enhanced the resolution resulting in separation of all cathinones and their positional and optical isomers. CONCLUSIONS: A novel CE/MS technique has been introduced that combines low-flow sheathless CE/MS, with HS-γ-CD and 15 mM (+)-18-C-6-TCA as the BGE for separation of cathinone derivatives as well as their positional and optical isomers.

Assessing the impact of synchrotron X-ray irradiation on proteinaceous specimens at macro and molecular levels.

Synchrotron radiation (SR) has become a preferred technique for the analysis of a wide range of archeological samples, artwork, and museum specimens. While SR is called a nondestructive technique, its effect on proteinaceous specimens has not been fully investigated at the molecular level. To investigate the molecular level effects of synchrotron X-ray on proteinaceous specimens, we propose a methodology where four variables are considered: (1) type of specimen: samples ranging from amino acids to proteinaceous objects such as silk, wool, parchment, and rabbit skin glue were irradiated; (2) synchrotron X-ray energy; (3) beam intensity; (4) irradiation time. Irradiated specimens were examined for both macroscopic and molecular effects. At macroscopic levels, color change, brittleness, and solubility enhancement were observed for several samples within 100 s of irradiation. At molecular levels, the method allowed one to quantify significant amino acid modifications. Aspartic acid (Asp), wool, parchment, and rabbit skin glue showed a significant increase in Asp racemization upon increasing irradiation time with rabbit skin glue showing the greatest increase in d-Asp formation. In contrast, Asp in silk, pure cystine (dimer of cysteine), and asparagine (Asn) did not show signs of racemization at the irradiation times studied; however, the latter two compounds showed significant signs of decomposition. Parchment and rabbit skin glue exhibited racemization of Asp, as well as racemization of isoleucine (Ile) and phenylalanine (Phe) after 100 s of irradiation with a focused beam. Under the experimental conditions and sample type and dimensions used here, more change was observed for focused and low energy (8 keV) beams than unfocused or higher energy (22 keV) beams. These results allow quantification of the change induced at the molecular level on proteinaceous specimens by synchrotron X-ray radiation and help to define accurate thresholds to minimize the probability of damage occurring to cultural heritage specimens. For most samples, damage was usually observed in the 1-10 s time scale, which is about an order of magnitude longer than SR studies of cultural heritage under X-ray fluorescence (XRF) mode; however, it is consistent with the duration of X-ray absorption spectroscopy (XAS) and microcomputed tomography (µCT) measurements.

Ultrafast capillary electrophoresis/mass spectrometry with adjustable porous tip for a rapid analysis of protein digest in about a minute.

RATIONALE: This paper introduces a novel ultrafast capillary electrophoresis (UFCE) mass spectrometry (MS) method comprised of a short (< 20 cm) and narrow (≤ 5 µm) capillary, with an integrated porous segment at the terminus of the capillary, for sheathless interfacing of CE to MS. The sheathless nature of the interface minimizes dead volume which is particularly important for ultrafast CE because of the small diameters of the capillaries. METHODS: The separation voltage across the capillary was set to produce an electric field of ≥1000 V/cm to provide an analysis time of approximately 1 min. For the analysis of peptides and protein digests, a background electrolyte containing 0.1% cationic polymer (polybrene or Poly-E) in 0.1% acetic acid in water was used to prevent analyte-wall interaction. RESULTS: Uniquely, the porous tip of the capillary is adjustable. This allows the porous tip to be pulled inside the sheath liquid for sample stacking during injection, or sample focusing in isoelectric focusing (IEF), or, alternatively, pushed outside of the sheath liquid for electrospray ionization (ESI). The adjustable interface preserves electrical conductivity during electrokinetic injection with reverse electro-osmotic flow. Therefore, pre-concentration of analytes at the capillary inlet is achieved without the need to disassemble the interface, improving the throughput. CONCLUSIONS: As a proof of concept, the application of UFCE-MS was demonstrated by the analysis of a peptide mixture and a protein digest in ~1 min.

Understanding irregular shell formation of Nautilus in aquaria: chemical composition and structural analysis.

Irregular shell formation and black lines on the outside of live chambered nautilus shells have been observed in all adult specimens at aquariums and zoos soon after the organisms enter aquaria. Black lines have also been observed in wild animals at sites of broken shell, but continued growth from that point returns to a normal, smooth structure. In contrast, rough irregular deposition of shell continues throughout residence in aquaria. The composition and reasons for deposition of the black material and mitigation of this irregular shell formation is the subject of the current study. A variety of analytical techniques were used, including stable isotope mass spectrometry (SI-MS), inductively coupled plasma mass spectrometry (ICP-MS), micro x-ray fluorescence (µXRF), X-ray diffraction (XRD), and scanning electron microscopy (SEM) based X-ray microanalysis. Results indicate that the black material contains excess amounts of copper, zinc, and bromine which are unrelated to the Nautilus diet. The combination of these elements and proteins plays an important role in shell formation, growth, and strengthening. Further study will be needed to compare the proteomics of the shell under aquaria versus natural wild environments. The question remains as to whether the occurrence of the black lines indicates normal healing followed by growth irregularities that are caused by stress from chemical or environmental conditions. In this paper we begin to address this question by examining elemental and isotopic differences of Nautilus diet and salt water. The atomic composition and light stable isotopic ratios of the Nautilus shell formed in aquaria verses wild conditions are presented.

Dating human bone: is racemization dating species-specific?

Our recently developed dating technique based on the racemization rate of aspartic acid was applied to dating human bone, as well as that of other mammals, utilizing capillary electrophoresis mass spectrometry. First, several well-dated (mostly (14)C-dated and with strong archeological evidence) human bones ranging in age from 150 to ~10,000 years were used to develop a calibration curve for human bone. The D/L ratio of aspartic acid for these specimens ranged from 2.4% to ~10%, with a correlation coefficient of better than 0.99, indicating a strong linear relationship between the d/l ratio of aspartic acid and the age of the specimens. This calibration curve can now be used to date human archeological specimens of unknown age, up to ~10,000 years. However, when the technique was applied to well-dated mixed species of larger mammal bones such as bison, whale, llama, etc., the calibration curve showed a slower rate of racemization with a lower correlation (0.88). As additional large mammal bones with less certain age (i.e., using archeological evidence alone with no (14)C-dating) were dated the correlation coefficient decreased to 0.70. The correlation coefficient decreased further to 0.58 when the racemization data from all mammals (including human) were added to the calibration curve, indicating the importance of using well-dated, species-specific specimens for forming a calibration curve. This conclusion is consistent with our previously published calibration curve for a single species of silk (Bombyx mori), which followed the expected reversible first-order kinetics. These results support species specificity of amino acid racemization dating.

Dating silk by capillary electrophoresis mass spectrometry.

A new capillary electrophoresis mass spectrometry (CE-MS) technique is introduced for age estimation of silk textiles based on amino acid racemization rates. With an L to D conversion half-life of ~2500 years for silk (B. mori) aspartic acid, the technique is capable of dating silk textiles ranging in age from several decades to a few-thousand-years-old. Analysis required only ~100 µg or less of silk fiber. Except for a 2 h acid hydrolysis at 110 °C, no other sample preparation is required. The CE-MS analysis takes ~20 min, consumes only nanoliters of the amino acid mixture, and provides both amino acid composition profiles and D/L ratios for ~11 amino acids.

Age estimation of museum wool textiles from Ovis aries using deamidation rates utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Cultural heritage contains a large number of precious proteinaceous specimens, such as wool and silk textiles, leather objects, paper, paint, coatings, binders (and associated adhesives), etc. To minimize the degradation of and to preserve these artifacts, it is desirable to understand the fundamental factors that cause their degradation, to identify the deterioration markers that determine their degradation stage and their age, and to use technologies that can provide this information rapidly while consuming a minimal amount of sample. There are several forces that cause protein degradation, including amino acid racemization, protein deamidation, and protein truncation. The purpose of this paper is to study protein deamidation using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for high-throughput dating of museums wool specimens. For proof of concept, several well-dated sheep's wool textiles from museum collections were analyzed. For wool samples aged from the present to ~400 years ago, the deamidation of two asparagine-containing peptides obtained from the tryptic digest of sheep wool were found to behave linearly in time, indicating that they could act as a potential biomarker of aging for wool samples.

High-throughput analysis using gated multi-inlet mass spectrometry.

Gated multi-inlet mass spectrometry is introduced for high-throughput chemical analysis. In this design, multiple high-pressure liquid chromatography (HPLC) columns or capillary electrophoresis (CE) capillaries are attached to multiple electrosprayers (one for each column or capillary) that spray toward a gated multi-inlet time-of-flight mass spectrometer (TOF-MS). Although all of the sprayers are spraying continuously, only one inlet is exposed at any given time for a specific duration set by the MS data system. The gated multi-sprayer, multi-inlet design significantly enhances the performance of the multi-ESI, multi-inlet TOF-MS with minimal cost and reduces analysis time. The gated multi-sprayer, multi-inlet design was applied to the investigation of column-to-column reproducibility of multiple HPLCs using a peptide mixture and to the simultaneous analysis of four protein digests. In addition, it was applied to the analysis of peptide mixtures using eight CE capillaries. The gated multi-inlet MS has several advantages compared to our previous non-gated multi-inlet MS. For example, because only one inlet is open at one time, the original manufacturer's inlet inner diameter and pumping system can be used, which enhances the sensitivity of detection for each inlet and minimizes the manufacturing cost. In addition, the number of inlets can be increased as desired. The maximum number of liquid streams that can be concurrently analyzed is limited by: (1) the number of inlets, (2) the chromatographic (electrophoretic) peak width, and (3) how fast the gate can move from one position to the next.

Metal displacement and stoichiometry of protein-metal complexes under native conditions using capillary electrophoresis/mass spectrometry.

Increases in the study of protein-metal complexes, as well as in metal displacement in protein-metal complexes under native conditions for optimum catalytic properties in drug research and catalyst design, demands a separation/detection technology that can accurately measure metal displacement and stoichiometry in protein-metal complexes. Both nuclear magnetic resonance (NMR) and X-ray diffraction techniques have been used for this purpose; however, these techniques lack sensitivity. Electrospray ionization mass spectrometry (ESI-MS) using direct infusion offers higher sensitivity than the former techniques and provides molecular distribution of various protein-metal complexes. However, since protein-metal complexes under native conditions usually are dissolved in salt solutions, their direct ESI-MS analysis requires off-line sample clean-up prior to MS analysis to avoid sample suppression during ESI. Moreover, direct infusion of the salty solution promotes non-specific salt adduct formation by the protein-metal complexes under ESI-MS, which complicates the identification and stoichiometry measurements of the protein-metal complexes. Because of the high mass of protein-metal complexes and lack of sufficient resolution by most mass spectrometers to separate non-specific from specific metal-protein complexes, accurate protein-metal stoichiometry measurements require some form of sample clean up prior to ESI-MS analysis. In this study, we demonstrate that capillary electrophoresis/electrospray ionization in conjunction with a medium-resolution (approximately 10,000) mass spectrometer is an efficient and fast method for the measurement of the stoichiometry of the protein-metal complexes under physiological conditions (pH approximately 7). The metal displacement of Co(2+) to Cd(2+), two metal ions necessary for activation in the monomeric AHL lactonase produced by B. thuringiensis, has been used as a proof of concept.

Analysis of major protein-protein and protein-metal complexes of erythrocytes directly from cell lysate utilizing capillary electrophoresis mass spectrometry.

The separation and detection of the major protein-protein and protein-metal complexes of erythrocytes directly from cell lysate under native conditions has been accomplished for the first time using capillary electrophoresis electrospray ionization-mass spectrometry (CE/ESI-MS). All three major protein-protein and protein-metal complexes in human red blood cells (RBCs) with a concentration dynamic range of approximately 3 orders of magnitude were successfully detected. Intact complexes detected in lysed RBCs included carbonic anhydrase II (CAII-Zn at approximately 0.8 amol/cell) complexed with its zinc cofactor, carbonic anhydrase I (CAI-Zn at approximately 7 amol/cell) complexed with its zinc cofactor, and hemoglobin A (Hb-tetramer at approximately 450 amol/cell)a tetramer formed by two alpha-beta-subunits and four heme groups. The average molecular weights measured for these complexes were consistent with their theoretical values within 0.01% mass accuracy. The use of Polybrene as a self-coating reagent in conjunction with ammonium acetate at pH approximately 7.4, narrow capillary for high separation efficiency, and forward polarity CE to avoid acid production at the tip of the capillary were overriding experimental factors for successful analysis of protein complexes. Diluting the lysed blood sample in ammonium acetate for a minimum of 6 h before injecting the sample into the CE was essential for obtaining the mass accuracy consistent with their theoretical average molecular weights. At physiological pH, the mass spectrum of the electrophoretic peak of Hb-tetramer included a small amount of the monomers and Hb-dimer. The migration time and peak profile of these species were almost identical to that of the tetramer, indicating that they are formed from decomposition of the Hb-tetramer during the ESI process. A separate electrophoretic peak for the Hb-dimer was only detected when the pH of the BGE was lowered from 7.4 to approximately 6.6. Running CE in forward polarity mode was essential for detection of the intact Hb-tetramer as well as CAI-Zn and CAII-Zn complexes. Under forward polarity mode, CE outlet/ESI shared electrode acts as the cathode of the CE circuit and the anode (positive voltage for positive ions) of the ESI circuit, thereby maintaining approximately neutral pH at the CE outlet/ESI electrode. In addition, under forward polarity mode, CAII-Zn and CAI-Zn migrated ahead of Hb-tetramer, avoiding being masked by 562x and 64x, respectively, molar excess of Hb-tetramer.

Toward Confirmatory On-Site Real-Time Detection of Emerging Drugs Using Portable Ultrafast Capillary Electrophoresis Mass Spectrometry.

Currently, law enforcement agencies rely upon presumptive tests such as color tests (or spot tests) for on-site, real-time identification of forensic evidence, such as controlled substances. These tests are simple and easy to use and require no instrumentation. However, they are unreliable and have a large false positive rate. On the other hand, confirmatory tests are done in analytical laboratories using sophisticated instrumentation by expert analysts, and have lower false positive rates. However, they are bulky and impractical for on-site real-time analysis. To provide more accurate identification of forensic evidence on-site, in real-time, it is important to develop portable confirmatory instrumentation using information-rich technologies. Moreover, because the analysis of controlled substances could be complicated by the existence of various isomers (including optical isomers) it is desirable that the portable instruments have the capability to separate structural and optical isomers of the controlled substances, because scheduling is some times dependent upon which isomer is present. To this end, we have developed a portable ultrafast capillary electrophoresis (UFCE) system for the separation of controlled substances and their structural and optical isomers. The UFCE instrument has an integrated porous tip for facile interfacing with electrospray ionization mass spectrometry. The technique has been successfully applied to the analysis of mixtures of several controlled substances such as amphetamines, cathinones, nor-mephedrone, and pregabalin and their optical isomers in about a minute.

A comparison between DART-MS and DSA-MS in the forensic analysis of writing inks.

Ambient ionization mass spectrometry is gaining momentum in forensic science laboratories because of its high speed of analysis, minimal sample preparation, and information-rich results. One such application of ambient ionization methodology includes the analysis of writing inks from questioned documents where colorants of interest may not be soluble in common solvents, rendering thin layer chromatography (TLC) and separation-mass spectrometry methods such as LC/MS (-MS) impractical. Ambient ionization mass spectrometry uses a variety of ionization techniques such as penning ionization in Direct Analysis in Real Time (DART), and atmospheric pressure chemical ionization in Direct Sample Analysis (DSA), and electrospray ionization in Desorption Electrospray Ionization (DESI). In this manuscript, two of the commonly used ambient ionization techniques are compared: Perkin Elmer DSA-MS and IonSense DART in conjunction with a JEOL AccuTOF MS. Both technologies were equally successful in analyzing writing inks and produced similar spectra. DSA-MS produced less background signal likely because of its closed source configuration; however, the open source configuration of DART-MS provided more flexibility for sample positioning for optimum sensitivity and thereby allowing smaller piece of paper containing writing ink to be analyzed. Under these conditions, the minimum sample required for DART-MS was 1mm strokes of ink on paper, whereas DSA-MS required a minimum of 3mm. Moreover, both techniques showed comparable repeatability. Evaluation of the analytical figures of merit, including sensitivity, linear dynamic range, and repeatability, for DSA-MS and DART-MS analysis is provided. To the forensic context of the technique, DART-MS was applied to the analysis of United States Secret Service ink samples directly on a sampling mesh, and the results were compared with DSA-MS of the same inks on paper. Unlike analysis using separation mass spectrometry, which requires sample preparation, both DART-MS and DSA-MS successfully analyzed writing inks with minimal sample preparation.

Protein extraction from human anagen head hairs 1-millimeter or less in total length.

A simple method for extracting protein from human anagen (i.e., actively growing hair stage) head hairs was developed in this study for cases of limited sample availability and/or studies of specific micro-features within a hair. The distinct feature segments of the hair from one donor were divided lengthwise (i.e., each of ∼200-400 µm) and then pooled for three individual hairs to form a total of eight composite hair samples (i.e., each of ∼1 mm or less in total length). The proteins were extracted, digested using trypsin, and characterized via nano-flow liquid chromatography tandem-mass spectrometry (nLC-MS/MS). A total of 63 proteins were identified from all eight protein samples analyzed of which 60% were keratin and keratin-associated proteins. The major hair keratins identified are consistent with previous studies using fluorescence in situ hybridization and nLC-MS/MS while requiring over 400-8000-fold less sample. The protein extraction method from micro-sized human head hairs described in this study will enable proteomic analysis of biological evidence for cases of limited sample availability and will complement hair research. For example, research seeking to develop alternative non-DNA based techniques for comparing questioned to known hairs, and understanding the biochemistry of hair decomposition.

Simplifying capillary electrophoresis-mass spectrometry operation: eliminating capillary derivatization by using self-coating background electrolytes.

To simplify capillary electrophoresis-mass spectrometry (CE-MS) operation, a background electrolyte (BGE) containing a polymer additive is introduced that allows the analysis of peptides and protein mixtures in underivatized fused-silica capillaries without any pretreatment, thereby increasing throughput. The most important characteristic of these polymer additives is that they do not significantly suppress the signals of the proteins and peptides under electrospray ionization, thereby allowing them to be used as an additive to common BGEs that are used for CE-MS analysis of peptide and protein mixtures. In addition, because the fused-silica capillary inner wall is continuously coated with the polymer additive, migration irreproducibility, due to the degradation of the capillary inner wall coating, under CE-MS is minimized. High sensitivity of detection, migration reproducibility, and ease of fabrication allow CE-MS analyses that require long analysis time, such as (CE-MS/MS)n, to be performed with ease. The utility of this background electrolyte has been demonstrated for the analysis of complex protein digests and intact proteins.

Clustering of childhood cancer in the inner city of Tehran metropolitan area: a GIS-based analysis.

The aim of this study was both to map the childhood cancer incidence in the districts of Tehran metropolitan area and to explore possible clustering of cancer cases in the diverse environments of this area. All incidence cases of childhood cancers (age group under 15 years) belonging to the 22 districts of Tehran metropolis and occurring during the period of 1998 till 2002 were ascertained from three sources. Each case's place of residency was geo-referenced. The scan statistics cluster detecting technique was used to evaluate clustering of cases throughout Tehran. The overall incidence rate (IR) of childhood cancer was 176.3/1,000,000 children under 15 years of age. The lowest IR among both boys and girls was observed in district 22 (69.4/1,000,000) and the highest was observed in district 6 (242.09/1,000,000). The detection of clusters was performed for all cancer sites. All the cancer sites combined category showed clustering in the districts 7, 13, 8, 6, 3, 14, 12, 11, and 4. For this category, the clustering likelihood was marginally statistically significant (p-value=0.056), with an overall relative risk of 1.30. No statistically significant patterns of clustering were detected for other categories.

Portable, Battery Operated Capillary Electrophoresis with Optical Isomer Resolution Integrated with Ionization Source for Mass Spectrometry.

We introduce a battery operated capillary electrophoresis electrospray ionization (CE/ESI) source for mass spectrometry with optical isomer separation capability. The source fits in front of low or high resolution mass spectrometers similar to a nanospray source with about the same weight and size. The source has two high voltage power supplies (±25 kV HVPS) capable of operating in forward or reverse polarity modes and powered by a 12 V rechargeable lithium ion battery with operation time of ~10 h. In ultrafast CE mode, in which short narrow capillaries (≤15 µm i.d., 15-25 cm long) and field gradients ≥1000 V/cm are used, peak widths at the base are <1 s wide. Under these conditions, the source provides high resolution separation, including optical isomer resolution in ~1 min. Using a low resolution mass spectrometer (LTQ Velos) with a scan time of 0.07 s/scan, baseline separation of amino acids and their optical isomers were achieved in ~1 min. Moreover, bovine serum albumin (BSA) was analyzed in ~1 min with 56% coverage using the data-dependent MS/MS. Using a high resolution mass spectrometer (Thermo Orbitrap Elite) with 15,000 resolution, the fastest scan time achieved was 0.15 s, which was adequate for CE-MS analysis when optical isomer separation is not required or when the optical isomers were well separated. Figures of merit including a detection limit of 2 fmol and linear dynamic range of two orders of magnitude were achieved for amino acids.