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It is frequently recounted that shortly before Michelangelo died, the eighty-eight-year-old artist sat before a fire and burned many drawings. Critical examination reveals various motivations for s...

Recent reports have demonstrated that genetically variant peptides derived from human hair shaft proteins can be used to differentiate individuals of different biogeographic origins. We report a method involving direct extraction of hair shaft proteins more sensitive than previously published methods regarding GVP detection. It involves one step for protein extraction and was found to provide reproducible results. A detailed proteomic analysis of this data is presented that led to the following four results: (i) A peptide spectral library was created and made available for download. It contains all identified peptides from this work, including GVPs that, when appropriately expanded with diverse hair‐derived peptides, can provide a routine, reliable, and sensitive means of analyzing hair digests; (ii) an analysis of artifact peptides arising from side reactions is also made using a new method for finding unexpected modifications; (iii) detailed analysis of the gel‐based method employed clearly shows the high degree of cross‐linking or protein association involved in hair digestion, with major GVPs eluting over a wide range of high molecular weights while others apparently arise from distinct non‐cross‐linked proteins; and (v) finally, we show that some of the specific GVP identifications depend on the sample preparation method.

This work presents methods for identifying and then creating a mass spectral library for disulfide-linked peptides originating from the NISTmAb, a reference material of the humanized IgG1k monoclonal antibody (RM 8671). Analyses involved both partially reduced and non-reduced samples under neutral and weakly basic conditions followed by nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS). Spectra of peptides containing disulfide bonds are identified by both MS1 ion and MS2 fragment ion data in order to completely map all the disulfide linkages in the NISTmAb. This led to the detection of 383 distinct disulfide-linked peptide ions, arising from fully tryptic cleavage, missed cleavage, irregular cleavage, complex Met/Trp oxidation mixtures, and metal adducts. Fragmentation features of disulfide bonds under low-energy collision dissociation were examined. These include (1) peptide bond cleavage leaving disulfide bonds intact; (2) disulfide bond cleavage, often leading to extensive fragmentation; and (3) double cleavage products resulting from breakages of two peptide bonds or both peptide and disulfide bonds. Automated annotation of various complex MS/MS fragments enabled the identification of disulfide-linked peptides with high confidence. Peptides containing each of the nine native disulfide bonds were identified along with 86 additional disulfide linkages arising from disulfide bond shuffling. The presence of shuffled disulfides was nearly completely abrogated by refining digest conditions. A curated spectral library of 702 disulfide-linked peptide spectra was created from this analysis and is publicly available for free download. Since all IgG1 antibodies have the same constant regions, the resulting library can be used as a tool for facile identification of "hard-to-find" disulfide-bonded peptides. Moreover, we show that one may identify such peptides originating from IgG1 proteins in human serum, thereby serving as a means of monitoring the completeness of protein reduction in proteomics studies. Data are available via ProteomeXchange with identifier PXD023358.

We describe the creation of a mass spectral library of acylcarnitines and conjugated acylcarnitines from the LC-MS/MS analysis of six NIST urine reference materials. To recognize acylcarnitines, we conducted in-depth analyses of fragmentation patterns of acylcarnitines and developed a set of rules, derived from spectra in the NIST17 Tandem MS Library and those identified in urine, using the newly developed hybrid search method. Acylcarnitine tandem spectra were annotated with fragments from carnitine and acyl moieties as well as neutral loss peaks from precursors. Consensus spectra were derived from spectra having similar retention time, fragmentation pattern, and the same precursor m/z and collision energy. The library contains 157 different precursor masses, 586 unique acylcarnitines, and 4 332 acylcarnitine consensus spectra. Furthermore, from spectra that partially satisfied the fragmentation rules of acylcarnitines, we identified 125 conjugated acylcarnitines represented by 987 consensus spectra, which appear to originate from Phase II biotransformation reactions. To our knowledge, this is the first report of conjugated acylcarnitines. The mass spectra provided by this work may be useful for clinical screening of acylcarnitines as well as for studying relationships among fragmentation patterns, collision energies, structures, and retention times of acylcarnitines. Further, these methods are extensible to other classes of metabolites.

This manuscript outlines a straight-forward procedure for generating a map of similarity between spectra of a set. When applied to a reference set of spectra for Type I fentanyl analogs (molecules differing from fentanyl by a single modification), the map illuminates clustering that is applicable to automated structure assignment of unidentified molecules. An open-source software implementation that generates mass spectral similarity mappings of unknowns against a library of Type I fentanyl analog spectra is available at http://github.com/asm3-nist/FentanylClassifier.

RATIONALE The metabolite profiling of a NIST plasma Standard Reference Material (SRM 1950) on different liquid chromatography/mass spectrometry (LC/MS) platforms showed significant differences. Although these findings suggest caution when interpreting metabolomics results, the degree of overlap of both profiles allowed us to use tandem mass spectral libraries of recurrent spectra to evaluate to what extent these results are transferable across platforms and to develop cross-platform chemical signatures. METHODS Non-targeted global metabolite profiles of SRM 1950 were obtained on different LC/MS platforms using reversed-phase chromatography and different chromatographic scales (conventional HPLC, UHPLC and nanoLC). The data processing and the metabolite differential analysis were carried out using publically available (XCMS), proprietary (Mass Profiler Professional) and in-house software (NIST pipeline). RESULTS Repeatability and intermediate precision showed that the non-targeted SRM 1950 profiling was highly reproducible when working on the same platform (relative standard deviation (RSD)

This essay is a preliminary sketch for a book that examines Michelangelo Buonarroti’s final eighteen years, from his appointment as architect of St. Peter’s until his death in 1564, that is, from age seventy-one to a few weeks shy of eighty-nine. This period represents nearly a quarter of his approximately seventy-five-year artistic career, yet it remains the least familiar segment of Michelangelo’s long life. It is paradoxical that in the final phase of his career, Michelangelo remained prodigiously creative and influential without being prolific — as he had been earlier in his career. His late life was concerned less with making things than with finding the courage and devotion to continue tasks that he knew he would never see to fruition, and this despite the loss of his closest friends, greatest patron, and his entire family.

A mass spectral library search algorithm that identifies compounds that differ from library compounds by a single ‘inert’ structural component is described. This algorithm, the Hybrid Similarity Search, generates a similarity score based on matching both fragment ions and neutral losses. It employs the parameter DeltaMass, defined as the mass difference between query and library compounds, to shift neutral loss peaks in the library spectrum to match corresponding neutral loss peaks in the query spectrum. When the spectra being compared differ by a single structural feature, these matching neutral loss peaks should contain that structural feature. This method extends the scope of the library to include spectra of ‘nearest-neighbor’ compounds that differ from library compounds by a single chemical moiety. Additionally, determination of the structural origin of the shifted peaks can aid in the determination of the chemical structure and fragmentation mechanism of the query compound. A variety of examples are presented, including the identification of designer drugs and chemical derivatives not present in the library.

A stochastic regression model is presented that separates signal from noise in chemical spectra. Spectra are decomposed into additive contributions from signal and from estimated noise. Numerical results on sample spectra are presented and suggest that this strategy offers an effective and computationally efficient framework for comprehensive noise estimation and analysis. From this analysis more effective methods of feature extraction in chemical spectra can be created.

A conceptually new approach for aligning chromatograms is introduced and applied to examples of metabolite identification in human blood plasma by liquid chromatography-mass spectrometry (LC-MS). A square-root representation of the chromatogram's derivative coupled with an extended Fisher-Rao metric enables the computation of relative differences between chromatograms. Minimization of these differences using a common dynamic programming algorithm brings the chromatograms into alignment. Application to a complex sample, National Institute of Standards and Technology (NIST) Standard Reference Material 1950, Metabolites in Human Plasma, analyzed by two different LC-MS methods having significantly different ranges of elution time is described.

A method to discover and correct errors in mass spectral libraries is described. Comparing across a set of highly curated reference libraries compounds that have the same chemical structure quickly identifies entries that are outliers. In cases where three or more entries for the same compound are compared, the outlier as determined by visual inspection was almost always found to contain the error. These errors were either in the spectrum itself or in the chemical descriptors that accompanied it. The method is demonstrated on finding errors in compounds of forensic interest in the NIST/EPA/NIH Mass Spectral Library. The target list of compounds checked was the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) mass spectral library. Some examples of errors found are described. A checklist of errors that curators should look for when performing inter-library comparisons is provided. Graphical Abstract ᅟ.

Non-targeted global metabolite profiles of Standard Reference Material (SRM) 1950 Metabolites in Human Plasma were obtained on five different liquid chromatography/mass spectrometry (LC/MS) instrument combinations using identically prepared samples. Different instruments rendered significantly different metabolite profiles, however, the results were highly reproducible when working with one instrument. A full description of the measurements and data analysis have been previously published [1]. Presented here are the raw data on which Ref. [1] is based. For a further description of SRM 1950 and the general analysis of its chemical composition, see Refs. [2] and [3].

Enzymes immobilized on solid supports are increasingly used for greener, more sustainable chemical transformation processes. Here, we used microreactors to study enzyme-catalyzed ring-opening polymerization of ε-caprolactone to polycaprolactone. A novel microreactor design enabled us to perform these heterogeneous reactions in continuous mode, in organic media, and at elevated temperatures. Using microreactors, we achieved faster polymerization and higher molecular mass compared to using batch reactors. While this study focused on polymerization reactions, it is evident that similar microreactor based platforms can readily be extended to other enzyme-based systems, for example, high-throughput screening of new enzymes and to precision measurements of new processes where continuous flow mode is preferred. This is the first reported demonstration of a solid supported enzyme-catalyzed polymerization reaction in continuous mode.

Recently it has been shown that the cobaltocenium cation, prepared by the laser ablation of a CoCp(CO) 2 /fullerene matrix, may react with alkanes and polyethylenes in the gas phase via a dehydrogenation reaction to produce [Co(Cp) 2 (alkadiene)] + ions without chain scission (W.E. Wallace, Chem. Commun. 2007, 4525–4527). To better understand these experimental observations, density functional calculations were used to obtain the gas phase binding energies and molecular structures of cobaltocenium, Co(Cp) 2 + , with 1,3-butadiene, 2,4-hexadiene, and 2,3-hexadiene. Calculations were conducted for both cis and trans molecular configurations, in both singlet and triplet electronic states, and with a variety of cyclopentadienyl hapticities. For 1,3-butadiene the 18-electron rule would predict a [Co(η 3 -Cp) 2 (η 4 -1,3-butadiene)] + , however, the lowest energy structure, [Co(η 5 -Cp) 2 (1,3-butadiene)] + , has a higher than expected cyclopentadienyl hapticity. In this structure a distance of nearly 0.5 nm between the metal core and the butadiene ligand leads to very little electron sharing. Thus the detected ions are better described as non-covalent ion–molecule complexes. In turn, the lack of orbital overlap leads to a low enthalpy giving the cis -butadiene complex a −13.0 kJ/mol binding energy and the trans -butadiene binding a −3.8 kJ/mol binding energy. These low binding energies lead to low levels of charged alkanes in the reactive ion formation process in agreement with experimental observations.

A method is presented to test whether the conversion of the mass spectrum of a polydisperse analyte to its molecular mass distribution is quantitative. Mixtures of samples with different average molecular masses, coupled with a Taylor's expansion mathematical formalism, were used to ascertain the reliability of molecular mass distributions derived from mass spectra. Additionally, the method describes how the molecular mass distributions may be corrected if the degree of mass bias is within certain defined limits. This method was demonstrated on polydisperse samples of C(60) fullerenes functionalized with ethylpyrrolidine groups measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; however, it is applicable to any polydisperse analyte and mass spectrometric method as long as spectrum resolution allows individual oligomers to be identified. Mass spectra of the derivatized fullerenes taken in positive ion mode were shown to give an accurate measurement of the molecular mass distribution while those taken in negative ion mode were not. Differences in the mechanisms for ion formation are used to explain the discrepancy. Official contribution of the National Institute of Standards and Technology; not subject to copyright in the United States of America.