The role of mass spectrometry and related techniques in the analysis of extractable and leachable chemicals.

In addition to degradation products, impurities, and exogenous contaminants, industries such as pharmaceutical, food, and others must concern themselves with leachables. These chemicals can derive from containers and closures or migrate from labels or secondary containers and packaging to make their way into products. Identification and quantification of extractables (potential leachables) and leachables, typically trace level analytes, is a regulatory expectation intended to ensure consumer safety and product fidelity. Mass spectrometry and related techniques have played a significant role in the analysis of extractables and leachables (E&L). This review provides an overview of how mass spectrometry is used for E&L studies, primarily in the context of the pharmaceutical industry. This review includes work flows, examples of how identification and quantification is done, and the importance of orthogonal data from several different detectors. E&L analyses are driven by the need for consumer safety. These studies are expected to expand in existing areas (e.g., food, textiles, toys, etc.) and into new, currently unregulated product areas. Thus, this topic is of interest to audiences beyond just the pharmaceutical and health care industries. Finally, the potential of universal detector approaches used in other areas is suggested as an opportunity to drive E&L research progress in this arguably understudied, under-published realm.

Yeast Trm7 interacts with distinct proteins for critical modifications of the tRNAPhe anticodon loop.

Post-transcriptional modification of the tRNA anticodon loop is critical for translation. Yeast Trm7 is required for 2'-O-methylation of C(32) and N(34) of tRNA(Phe), tRNA(Trp), and tRNA(Leu(UAA)) to form Cm(32) and Nm(34), and trm7-Δ mutants have severe growth and translation defects, but the reasons for these defects are not known. We show here that overproduction of tRNA(Phe) suppresses the growth defect of trm7-Δ mutants, suggesting that the crucial biological role of Trm7 is the modification of tRNA(Phe). We also provide in vivo and in vitro evidence that Trm7 interacts with ORF YMR259c (now named Trm732) for 2'-O-methylation of C(32), and with Rtt10 (named Trm734) for 2'-O-methylation of N(34) of substrate tRNAs and provide evidence for a complex circuitry of anticodon loop modification of tRNA(Phe), in which formation of Cm(32) and Gm(34) drives modification of m(1)G(37) (1-methylguanosine) to yW (wyebutosine). Further genetic analysis shows that the slow growth of trm7-Δ mutants is due to the lack of both Cm(32) and Nm(34), and the accompanying loss of yW, because trm732-Δ trm734-Δ mutants phenocopy trm7-Δ mutants, whereas each single mutant is healthy; nonetheless, TRM732 and TRM734 each have distinct roles, since mutations in these genes have different genetic interactions with trm1-Δ mutants, which lack m(2,2)G(26) in their tRNAs. We speculate that 2'-O-methylation of the anticodon loop may be important throughout eukaryotes because of the widespread conservation of Trm7, Trm732, and Trm734 proteins, and the corresponding modifications, and because the putative human TRM7 ortholog FTSJ1 is implicated in nonsyndromic X-linked mental retardation.

Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14.

The modified nucleosides N(2)-methylguanosine and N(2)(2)-dimethylguanosine in transfer RNA occur at five positions in the D and anticodon arms, and at positions G6 and G7 in the acceptor stem. Trm1 and Trm11 enzymes are known to be responsible for several of the D/anticodon arm modifications, but methylases catalyzing post-transcriptional m(2)G synthesis in the acceptor stem are uncharacterized. Here, we report that the MJ0438 gene from Methanocaldococcus jannaschii encodes a novel S-adenosylmethionine-dependent methyltransferase, now identified as Trm14, which generates m(2)G at position 6 in tRNA(Cys). The 381 amino acid Trm14 protein possesses a canonical RNA recognition THUMP domain at the amino terminus, followed by a γ-class Rossmann fold amino-methyltransferase catalytic domain featuring the signature NPPY active site motif. Trm14 is associated with cluster of orthologous groups (COG) 0116, and most closely resembles the m(2)G10 tRNA methylase Trm11. Phylogenetic analysis reveals a canonical archaeal/bacterial evolutionary separation with 20-30% sequence identities between the two branches, but it is likely that the detailed functions of COG 0116 enzymes differ between the archaeal and bacterial domains. In the archaeal branch, the protein is found exclusively in thermophiles. More distantly related Trm14 homologs were also identified in eukaryotes known to possess the m(2)G6 tRNA modification.

Safety assessment scheme for menstrual cups and application for the evaluation of a menstrual cup comprised of medical grade silicone.

BACKGROUND: Ensuring menstrual cup safety is paramount, yet a menstrual cup safety assessment scheme is lacking. This paper presents a quadripartite scheme, showing how it can be applied. METHODS: The Tampax Menstrual Cup was evaluated in the safety assessment scheme: (1) Biocompatibility and chemical safety of cup constituents. Extractables were obtained under different use condition; exposure-based risk assessments (EBRA) were conducted for extractables exceeding thresholds of toxicological concern. (2) Physical impact to vaginal mucosa. After physical evaluations, the Tampax Cup and another cup were assessed in a randomised double-blinded, two-product, two-period cross-over clinical trial (65 women, mean age 34.2 years). (3) Impact to vaginal microbiota (in vitro mixed microflora assay and evaluation of vaginal swabs). (4) In vitro growth of Staphylococcus aureus and toxic shock syndrome toxin-1 (TSST-1) production. FINDINGS: Biocompatibility assessments and EBRA of cup constituents showed no safety concerns. In the randomised clinical trial, all potentially product-related adverse effects were mild, vaginal exams were unremarkable, no clinically relevant pH changes occurred, post-void residual urine volume with and without cup were similar, and self-reported measures of comfort along with reports of burning, itching and stinging between cups were comparable. Cup use had no effect on microbial growth in vitro or in the 62 subjects who completed the trial or on in vitro TSST-1 production. INTERPRETATION: The quadripartite safety assessment scheme allows evaluation of menstrual cup safety. The Tampax Cup is safe and well-tolerated upon intended use. As with all feminine hygiene products, post-market safety surveillance confirmed this conclusion. FUNDING: By Procter & Gamble.

Removal of 3'-phosphate group by bacterial alkaline phosphatase improves oligonucleotide sequence coverage of RNase digestion products analyzed by collision-induced dissociation mass spectrometry.

RNase mapping by nucleobase-specific endonucleases combined with liquid chromatography/tandem mass spectrometry (LC/MS/MS) is a powerful analytical method for characterizing ribonucleic acids (RNAs). Endonuclease digestion of RNA yields products that contain a 3'-terminal phosphate group. MS/MS via collision-induced dissociation (CID) of these digestion products on a linear ion trap generates fragmentation pathways that include the loss of phosphoric acid (-H(3)PO(4); -98 u), which does not provide information about the sequence of the digestion products and can reduce ion abundance from other pathways that provide sequence information. Here we investigate the use of bacterial alkaline phosphatase (BAP) after RNase digestion to remove the 3'-terminal phosphate from all RNase digestion products prior to LC/MS/MS analysis. RNase digestion products lacking the 3'-phosphate were found to produce CID spectra with more consistent, high-abundance c- and y-type fragment ions as well as significantly more a-Base and w-type ions than digestion products retaining the 3'-phosphate. In this manner, RNase mapping with LC/MS/MS can provide more complete RNA sequence information from fragment ions of higher abundance that are easier to interpret and identify.

Sequence analysis of peptide:oligonucleotide heteroconjugates by electron capture dissociation and electron transfer dissociation.

Mass spectrometry analysis of protein-nucleic acid cross-links is challenging due to the dramatically different chemical properties of the two components. Identifying specific sites of attachment between proteins and nucleic acids requires methods that enable sequencing of both the peptide and oligonucleotide component of the heteroconjugate cross-link. While collision-induced dissociation (CID) has previously been used for sequencing such heteroconjugates, CID generates fragmentation along the phosphodiester backbone of the oligonucleotide preferentially. The result is a reduction in peptide fragmentation within the heteroconjugate. In this work, we have examined the effectiveness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) for sequencing heteroconjugates. Both methods were found to yield preferential fragmentation of the peptide component of a peptide:oligonucleotide heteroconjugate, with minimal differences in sequence coverage between these two electron-induced dissociation methods. Sequence coverage was found to increase with increasing charge state of the heteroconjugate, but decreases with increasing size of the oligonucleotide component. To overcome potential intermolecular interactions between the two components of the heteroconjugate, supplemental activation with ETD was explored. The addition of a supplemental activation step was found to increase peptide sequence coverage over ETD alone, suggesting that electrostatic interactions between the peptide and oligonucleotide components are one limiting factor in sequence coverage by these two approaches. These results show that ECD/ETD methods can be used for the tandem mass spectrometry sequencing of peptide:oligonucleotide heteroconjugates, and these methods are complementary to existing CID methods already used for sequencing of protein-nucleic acid cross-links.