Rapid analysis of forensic-related samples using two ambient ionization techniques coupled to high-resolution mass spectrometers

RATIONALE: This paper highlights the versatility of interfacing two ambient ionization techniques, Laser Diode Thermal Desorption (LDTD) and Atmospheric Solids Analysis Probe (ASAP), to high-resolution mass spectrometers and demonstrate the method’s capability to rapidly generate high-quality data from multiple sample types with minimal, if any, sample preparation. METHODS: For ASAP-MS analysis of solid and liquid samples, the material was transferred to a capillary surface before being introduced into the mass spectrometer. For LDTD-MS analysis, samples were solvent extracted, spotted in a 96-well plate, and the solvent was evaporated before being introduced into the mass spectrometer. All analyses were performed using Atmospheric Pressure Chemical Ionization in positive mode. RESULTS: Seven consumer Spice packets were combined and analyzed by both ASAP and LDTD, which identified 11 synthetic cannabinoids/cathinones by full MS and MS/MS experiments. To further show the usefulness of these techniques, black tar heroin was analyzed, which resulted in the identification of heroin and its impurities (monoacetylmorphine, papaverine, and noscapine). These experiments were performed on the LTQ-Orbitrap to demonstrate the ability to perform both parallel and serial MS and MSn experiments. CONCLUSIONS: Interfacing LDTD and ASAP to high-resolution mass spectrometers allows for expeditious analysis of a wide range of samples, with minimal or no sample preparation. Both allow for rapid full scan, MS/MS, and/or MSn experiments from a single sample introduction

Peptide-binding motifs associated with MHC molecules common in Chinese rhesus macaques are analogous to those of human HLA supertypes and include HLA-B27-like alleles

Chinese rhesus macaques are of particular interest in SIV/HIV research as these animals have prolonged kinetics of disease progression to AIDS, compared to their Indian counterparts, suggesting that they may be a better model for HIV. Nevertheless, the specific mechanism(s) accounting for these kinetics remains unclear. The study of Major Histocompatibility Complex (MHC) molecules, including their MHC:peptide binding motifs, provides valuable information for measuring cellular immune responses and deciphering outcomes of infection and vaccine efficacy. In this study, we have provided detailed characterization of six prevalent Chinese rhesus macaque MHC class I alleles, yielding a combined phenotypic frequency of 29%. The peptide binding specificity of two of these alleles, Mamu-A2*01:02 and -B*010:01, as well as the previously characterized allele Mamu-B*003:01 (and Indian rhesus Mamu-B*003:01), was found to be analogous to that of alleles in the HLA-B27 supertype family. Specific alleles in the HLA-B27 supertype family, including HLA-B*27:05, have been associated with long-term non-progression to AIDS in humans. All six alleles characterized in the present study were found to have specificities analogous to HLA-supertype alleles. These data contribute to the concept that Chinese rhesus macaque MHC immunogenetics is more similar to HLA than their Indian rhesus macaque counterparts, and thereby warrant further studies to decipher the role of these alleles in the context of SIV infection

OGT (O-GlcNAC transferase) selectively modifies multiple residues unique to Lamin A

The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in LMNA cause over 12 diseases (‘laminopathies’). Lamins A and C are identical for their first 566 residues. However, they form separate filaments in vivo, with apparently distinct roles. We report that lamin A is β-O-linked N-acetylglucosamine-(O-GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified O-GlcNAc transferase (OGT) enzyme showed robust O-GlcNAcylation of recombinant mature lamin A tails (residues 385–646), with no detectable modification of lamin B1, lamin C, or ‘progerin’ (Δ50) tails. Using mass spectrometry, we identified 11 O-GlcNAc sites in a ‘sweet spot’ unique to lamin A, with up to seven sugars per peptide. Most sites were unpredicted by current algorithms. Double-mutant (S612A/T643A) lamin A tails were still robustly O-GlcNAc-modified at seven sites. By contrast, O-GlcNAcylation was undetectable on tails bearing deletion Δ50, which causes Hutchinson–Gilford progeria syndrome, and greatly reduced by deletion Δ35. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion Δ35, which does not remove the majority of identified O-GlcNAc sites, does remove potential OGT-association motifs (lamin A residues 622–625 and 639–645) homologous to that in mouse Tet1. These biochemical results are significant because they identify a novel molecular pathway that may profoundly influence lamin A function. The hypothesis that lamin A is selectively regulated by OGT warrants future testing in vivo, along with two predictions: genetic variants may contribute to disease by perturbing OGT-dependent regulation, and nutrient or other stresses might cause OGT to misregulate wildtype lamin A

The workflow of the virtual screening protocol for screening of similar drugs to abacavir.

<p>The workflow of the virtual screening protocol for screening of similar drugs to abacavir.</p

Effects of acyclovir (2 mg/mL) on the affinity of C-terminal residues for HLA-B*57:01.

<p>Values are represented as geometric mean with 95% CI of the fold difference between vehicle/acyclovir treatment. The experiment was run 6 times with each run performed in triplicates. Analyzed for statistical significance by column statistics; p < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p < 0.001). The most pronounced affinity increases for HLA-B*57:01 in the presence of 2 mg/mL of acyclovir were found for peptides with a cysteine, isoleucine and valine at the C-terminus.</p

PBMC from a healthy HLA-B*57:01 positive donor (Donor 1) where primed with abacavir at day 0, cultured for 14 days and then restimulated 1:10 with (A) HLA-B*57:01 single antigen line (C1R.B57), (B) with O/N abacavir treated C1R.B57 (C1R.B57.ABC) or (C) with O/N acyclovir treated C1R.B57 (C1R.B57.ACY).

<p>Antigen activated cells were detected by ICS for IFN-γ production and CD8+/ IFN-γ T-cells quantitated using flow cytometry. (D) PBMC from two healthy HLA-B*57:01 positive donors were either primed with abacavir (ABC primed), primed with acyclovir (ACY primed) or had no treatment (Control. PBMC were cultured for 14 days and then stimulated 1:10 with treated and untreated single antigen line, C1R.B57, as indicated.</p

Effects of abacavir and acyclovir on HLA-B*57:01 binding specificity.

<p>Specific peptides with a terminal valine that showed an increased affinity for HLA-B*57:01 in the presence of abacavir were tested. Values are represented as geometric mean with 95% CI of two independent runs in triplicates, analyzed for statistical significance by Mann-Whitney U test comparing log IC₅₀ values vs. vehicle; p < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p < 0.001).</p

Effects of abacavir and acyclovir on HLA-B*57:01 binding specificity.

<p>Specific peptides with a terminal isoleucine that showed an increased affinity for HLA-B*57:01 in the presence of abacavir were tested. Values are represented as geometric mean with 95% CI of two independent runs in triplicates, analyzed for statistical significance by Mann-Whitney U test comparing log IC₅₀ values vs. vehicle; p < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p < 0.001).</p

Acyclovir Has Low but Detectable Influence on HLA-B*57:01 Specificity without Inducing Hypersensitivity

<div><p>Immune mediated adverse drug reactions (IM-ADRs) remain a significant source of patient morbidity that have more recently been shown to be associated with specific class I and/or II human leukocyte antigen (HLA) alleles. Abacavir-induced hypersensitivity syndrome is a CD8⁺ T cell dependent IM-ADR that is exclusively mediated by HLA-B*57:01. We and others have previously shown that abacavir can occupy the floor of the peptide binding groove of HLA-B*57:01 molecules, increasing the affinity of certain self peptides resulting in an altered peptide-binding repertoire. Here, we have identified another drug, acyclovir, which appears to act in a similar fashion. As with abacavir, acyclovir showed a dose dependent increase in affinity for peptides with valine and isoleucine at their C-terminus. In agreement with the binding studies, HLA-B*57:01 peptide-elution studies performed in the presence of acyclovir revealed an increased number of endogenously bound peptides with a C-terminal isoleucine. Accordingly, we have hypothesized that acyclovir acts by the same mechanism as abacavir, although our data also suggest the overall effect is much smaller: the largest changes of peptide affinity for acyclovir were 2-5 fold, whereas for abacavir this effect was as much as 1000-fold. Unlike abacavir, acyclovir is not known to cause IM-ADRs. We conclude that the modest effect of acyclovir on HLA binding affinity in contrast to the large effect of abacavir is insufficient to trigger a hypersensitivity syndrome. We further support this by functional <i>in vitro</i> studies where acyclovir, unlike abacavir, was unable to produce an increase in IFN-γ upon expansion of HLA-B*57:01⁺ PBMCs from healthy donors. Using abacavir and acyclovir as examples we therefore propose an in vitro pre-clinical screening strategy, whereby thresholds can be applied to MHC-peptide binding assays to determine the likelihood that a drug could cause a clinically relevant IM-ADR.</p></div