Gunnar Schwarz

MeCAT—new iodoacetamide reagents for metal labeling of proteins and peptides

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.

DOTA based metal labels for protein quantification: a review

Today, quantitative data play a pivotal role in the understanding of biological processes. This is particularly true for the proteome: protein quantification always follows protein identification. To obtain useful and reliable quantitative data, rather sophisticated strategies using electrospray and MALDI mass spectrometry have been developed, which allow relative and sometimes even absolute quantification. All of those strategies have merits and limitations. In order to overcome some of these limits, methods based on the reliable and sensitive detection and quantification of heavy metals present in proteins using inductively coupled plasma (ICP)-MS have been reported. With specific labels carrying heavy metals, the applicability of ICP-MS has been extended to almost every protein. One of such covalently bound metal tags, allowing the quantification of low abundant proteins, uses 1,4,7,10-tetraazacyclododecane N,N′,N′′,N′′-tetraacetic acid (DOTA) chelate complexes carrying lanthanides as the metal core. In this review the scope and limitations of peptide and protein quantification will be addressed. The metal tags do not only provide low detection limits, but also due to the large number of different lanthanides and lanthanide isotopes, multiplexing capabilities and previously unknown accuracy based on inherently possible isotope dilution methods came into reach. The developed workflows, including electrophoretic and chromatographic separation and preconcentration techniques, will be addressed to allow a comparison with already established procedures.

High-Speed, High-Resolution, Multielemental Laser Ablation-Inductively Coupled Plasma-Time-of-Flight Mass Spectrometry Imaging: Part I. Instrumentation and Two-Dimensional Imaging of Geological Samples

Low-dispersion laser ablation (LA) has been combined with inductively coupled plasma-time-of-flight mass spectrometry (ICP-TOFMS) to provide full-spectrum elemental imaging at high lateral resolution and fast image-acquisition speeds. The low-dispersion LA cell reported here is capable of delivering 99% of the total LA signal within 9 ms, and the prototype TOFMS instrument enables simultaneous and representative determination of all elemental ions from these fast-transient ablation events. This fast ablated-aerosol transport eliminates the effects of pulse-to-pulse mixing at laser-pulse repetition rates up to 100 Hz. Additionally, by boosting the instantaneous concentration of LA aerosol into the ICP with the use of a low-dispersion ablation cell, signal-to-noise (S/N) ratios, and thus limits of detection (LODs), are improved for all measured isotopes; the lowest LODs are in the single digit parts per million for single-shot LA signal from a 10-μm diameter laser spot. Significantly, high-sensitivity, multielemental and single-shot-resolved detection enables the use of small LA spot sizes to improve lateral resolution and the development of single-shot quantitative imaging, while also maintaining fast image-acquisition speeds. Here, we demonstrate simultaneous elemental imaging of major and minor constituents in an Opalinus clay-rock sample at a 1.5 μm laser-spot diameter and quantitative imaging of a multidomain Pallasite meteorite at a 10 μm LA-spot size.

High-Speed, High-Resolution, Multielemental LA-ICP-TOFMS Imaging: Part II. Critical Evaluation of Quantitative Three-Dimensional Imaging of Major, Minor, and Trace Elements in Geological Samples

Here we describe the capabilities of laser-ablation coupled to inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) for high-speed, high-resolution, quantitative three-dimensional (3D) multielemental imaging. The basic operating principles of this instrumental setup and a verification of 3D quantitative elemental imaging are provided. To demonstrate the potential of 3D LA-ICP-TOFMS imaging, high-resolution multielement images of a cesium-infiltrated Opalinus clay rock were recorded using LA with a laser-spot diameter of 5 μm coupled to ICP-TOFMS. Quantification of elements ablated from each individual laser pulse was carried out by 100% mass normalization, and the 3D elemental concentration images generated match well with the expected distribution of elements. After laser-ablation imaging, the sample surface morphology was investigated using confocal microscopy, which showed substantial surface roughness and evidence of matrix-dependent ablation yields. Depth assignment based on ablation yields from heterogeneous materials, such as Opalinus clay rock, will remain a challenge for 3D LA-ICPMS imaging. Nevertheless, this study demonstrates quantitative 3D multielemental imaging of geological samples at a considerably higher image-acquisition speed than previously reported, while also offering high spatial resolution and simultaneous multielemental detection.

Comparison of the fragmentation behavior of differentially metal‐coded affinity tag (MeCAT)‐labeled peptides

Mass spectrometry (MS) became a pivotal technique in the still growing field of quantitative proteomics, since system biology needs quantitative information. This development is not only driven by newly arising demands, but from the availability of modern instrumentation and new quantitative methods. Thus, techniques like isotope-coded affinity tags, isobaric tag for relative and absolute quantification and stable isotope labeling with amino acids in cell culture have been used routinely in many laboratories for years. However, only a few of the methods in quantitative proteomics offer absolute quantification. Most approaches only deliver relative quantification data by direct comparison of two or more samples. In order to measure absolute quantities without the need for structurally similar or identical standards, we introduced metal-coded affinity tagging (MeCAT) for quantitative proteomics. Based upon a covalent label for proteins and peptides with a metal chelate complex (1,4,7,10-tetraazacyclododecane, DOTA) harboring a lanthanide ion, MeCAT directly enables the determination of protein and peptide amounts via inductively coupled plasma MS of the incorporated lanthanide ion. Additionally, we recently introduced a new improved reagent MeCAT-IA carrying an iodoacetamide moiety, which shows distinct advantages over the originally applied MeCAT-Mal with maleinimide reactivity. These advantages include the absence of diastereomer formation during labeling and an increased labeling yield that results in the use of lower amounts of MeCAT-IA for complete labeling of proteins and peptides. Furthermore, labeling with MeCAT-IA is also faster in comparison to MeCAT-Mal. While absolute quantification is possible down to very low amounts with the MeCAT methodology, quantification of a protein also depends on protein identification, and thus, unambiguous identification of labeled peptides is essential for an easy and reliable approach. When studying the fragmentation behavior of MeCAT-Mal-labeled peptides, we could demonstrate that MeCAT-Mal labeling does not hamper typical peptide fragmentation and thus peptide and protein identification. Here, we report the extension of this work with focus on the fragmentation behavior of MeCAT-IA-labeled peptides under different activation conditions (collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD)). Based on these experiments, we discuss differences in fragmentation behavior to unlabeled peptides, alkylated peptides and influences of the lanthanide ions within the MeCAT reagent. Finally, we have investigated the utilization of automated data base search algorithms for the identification of MeCAT-labeled peptides. By using peptides resulting from a trypsin digest of BSA, we were able to study the fragmentation behavior of a wide range

Capabilities of laser ablation inductively coupled plasma time-of-flight mass spectrometry

In this paper, we characterize an inductively coupled plasma time-of-flight mass spectrometry (ICP-TOFMS) instrument (icpTOF, TOFWERK AG, Thun, Switzerland) in combination with laser-ablation sample introduction. Three sample introduction approaches for LA-based ICP-TOFMS analysis are described: (1) steady-state LA with a conventional high-dispersion LA cell, (2) single-pulse analysis with large spot sizes (44 μm diameter) using a low-dispersion LA cell, and (3) pulse-resolved, high-speed, high-resolution (5 μm spot sizes) elemental imaging with the same low-dispersion LA cell. These sample-introduction schemes span the range of approaches most interesting for users of LA-ICP-TOFMS, from routine bulk quantification, to low-sample-consumption trace-element analysis, to demanding elemental imaging applications. From steady-state signal intensities, element concentrations in NIST SRM 612 and USGS BCR-2G were quantified within the uncertainty range of the preferred values when NIST SRM 610 was used as the external reference material. Relative deviations were less than 10% in most cases. When using a 44 μm diameter spot and a laser repetition rate of 10 Hz, limits of detection (LODs) were in the single digit ng g−1 range for the most sensitive isotopes. Isotope-ratio precision was in the sub per mill regime and governed by counting statistics. Similar accuracies were also achieved in low-dispersion LA-ICP-TOFMS experiments, when NIST SRM 612 and USGS BCR-2G element concentrations were quantified using signal intensities from single 44 μm diameter laser pulses. LODs were in the tens of ng g−1 range for most sensitive isotopes resulting in absolute LODs in the tens of attograms. Capabilities of the icpTOF for elemental imaging are demonstrated with pulse-resolved multi-elemental imaging of a multi-phase geological thin section. With the low-dispersion LA cell and a spot diameter of 5 μm, aerosol plumes were confined to less than 10 ms, which allowed elemental imaging at a laser repetition rate of 100 Hz with minimized pulse-to-pulse mixing and an adjacent-pixel dynamic range of greater than 102. Quantitative results for elements of major, minor and trace concentrations were in agreement with bulk composition of individual regions, which had been determined via petrographic microscopy and high-dispersion laser ablation inductively coupled plasma quadrupole mass spectrometry (LA-ICPQMS). LODs were in the single digit μg g−1 range for most sensitive isotopes.