High Mass Measurement Accuracy Research Articles

Multiple-reflection time-of-flight mass spectrometry

Multiple-reflection time-of-flight mass spectrometry is an emerging technique that meets the challenges of mass spectrometry at current and future accelerator facilities for the research with exotic nuclei. Moreover, many new applications in analytical mass spectrometry appear on the horizon. In a multiple-reflection time-of-flight mass spectrometer (MR-TOF-MS) the analyzer is traversed many times by the ions, extending the flight path by several orders of magnitude over conventional time-of-flight mass spectrometers (TOF-MS). MR-TOF-MS thus allow to achieve a very high mass resolving power (>100,000) in a compact device. They combine the advantages of conventional TOF-MS, viz. extremely short measurement times (∼ ms), a large mass range, very high sensitivity and non-scanning operation, with very high mass resolving power and accuracy. MR-TOF-MS can serve as devices for high-accuracy mass measurements of very short-lived nuclei, as high-resolution mass separators, and as broadband mass spectrometers for diagnostic purposes. In this article, an overview of MR-TOF-MS developments for the research with short-lived nuclei is given. Different instrumental implementations are reviewed. Despite the same operation principle, different instrumental solutions exist, which give rise to significant differences in applicability and performance. A novel performance criterion for mass spectrometers for the research with exotic nuclei is presented, and a performance increase for MR-TOF-MS of at least one order of magnitude compared to the established technique of TOF-ICR is found. The MR-TOF-MS for the Low-Energy Branch of the Super-FRS at the Facility for Antiproton and Ion Research (FAIR), which has recently been commissioned at the FRS Ion Catcher at GSI, is presented in some detail. Applications of MR-TOF-MS at accelerator facilities as well as their scientific potential outside the field of nuclear physics are discussed.

Mass Recalibration of FT-ICR Mass Spectrometry Imaging Data Using the Average Frequency Shift of Ambient Ions

Achieving and maintaining high mass measurement accuracy (MMA) throughout a mass spectrometry imaging (MSI) experiment is vital to the identification of the observed ions. However, when using FTMS instruments, fluctuations in the total ion abundance at each pixel due to inherent biological variation in the tissue section can introduce space charge effects that systematically shift the observed mass. Herein we apply a recalibration based on the observed cyclotron frequency shift of ions found in the ambient laboratory environment, polydimethylcyclosiloxanes (PDMS). This calibration method is capable of achieving part per billion (ppb) mass accuracy with relatively high precision for an infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI dataset. Comparisons with previously published mass calibration approaches are also presented.

High mass accuracy and high mass resolving power FT-ICR secondary ion mass spectrometry for biological tissue imaging

Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the sub-micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for elemental formula assignment based on exact mass measurement. We have recently reported a secondary ion mass spectrometer with the combination of a C60 primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy, and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 μm spatial resolution (75 μm primary ion spot size) with mass resolving power (m/Δm(50%)) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with m/Δm(50%) > 3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 μm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging.

Performance evaluation of a dual linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer for proteomics research

A novel dual cell linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) and its performance characteristics are reported. A linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer has been modified to incorporate a LTQ-Velos mass spectrometer. This modified instrument features efficient ion accumulation and fast MS/MS acquisition capabilities of dual cell linear RF ion trap instruments coupled to the high mass accuracy, resolution, and dynamic range of a FT-ICR for improved proteomic coverage. The ion accumulation efficiency is demonstrated to be an order of magnitude greater than that observed with LTQ-FT Ultra instrumentation. The proteome coverage with yeast was shown to increase over the previous instrument generation by 50% (100% increase on the peptide level). In addition, many lower abundance level yeast proteins were only detected with this modified instrument. This novel configuration also enables beam type CID fragmentation using a dual cell RF ion trap mass spectrometer. This technique involves accelerating ions between traps while applying an elevated DC offset to one of the traps to accelerate ions and induce fragmentation. This instrument design may serve as a useful option for labs currently considering purchasing new instrumentation or upgrading existing instruments. A novel hybrid mass spectrometer that allows increased MS/MS acquisition rates with high mass measurement accuracy and new ion fragmentation methods greatly improves the number of proteins, posttranslational modifications and protein-protein interactions that can be identified from cells.

Characterization of disulfide linkages in recombinant human granulocyte‐colony stimulating factor

Recombinant human G granulocyte-colony stimulating factor (rhG-CSF) produced in Escherichia coli is a non-glycosylated polypeptide containing five cysteine residues. The reported major disulfide (S-S) linkages in mature human G-CSF are C36 -C42 and C64 -C74 , leaving C17 as a free cysteine, which could potentially result in S-S scrambling. The purpose of this work is to illustrate different mass spectrometry (MS) approaches for characterization of S-S linkages in therapeutic proteins including S-S scrambling using rhG-CSF as a model protein. Peptide mapping analysis of both non-reduced and reduced digests of rhG-CSF was performed to demonstrate the presence of S-S linked peptides and their corresponding reduced peptides. High mass accuracy measurements of these peptides provided the initial identifications of S-S linkages. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) were used to fragment these peptides in order to obtain further sequence information and identify S-S linkages. S-S linked peptides and their corresponding reduced peptides correlating with major S-S linkages were observed. Peptides that correlated with other S-S linkages as a result of S-S scrambling were also observed. Presence of the reported major S-S linkages in rhG-CSF was confirmed. S-S scrambling was also observed in which C18 was involved in S-S linkages and C37 , C65 or C75 were present as free cysteines. This study demonstrates the practical utility of combining different MS methods for characterization of S-S linkages in therapeutic proteins.

Chemical cross‐linkers for protein structure studies by mass spectrometry

The cross-linking approach combined with MS for protein structure determination is one of the most striking examples of multidisciplinary success. Indeed, it has become clear that the bottleneck of the method was the detection and the identification of low-abundance cross-linked peptides in complex mixtures. Sample treatment or chromatography separation partially addresses these issues. However, the main problem comes from over-represented unmodified peptides, which do not yield any structural information. A real breakthrough was provided by high mass accuracy measurement, because of the outstanding technical developments in MS. This improvement greatly simplified the identification of cross-linked peptides, reducing the possible combinations matching with an observed m/z value. In addition, the huge amount of data collected has to be processed with dedicated software whose role is to propose distance constraints or ideally a structural model of the protein. In addition to instrumentation and algorithms efficiency, significant efforts have been made to design new cross-linkers matching all the requirements in terms of reactivity and selectivity but also displaying probes or reactive systems facilitating the isolation, the detection of cross-links, or the interpretation of MS data. These chemical features are reviewed and commented on in the light of the more recent strategies.

Tandem mass spectrometry of nitric oxide and hydrogen sulfide releasing aspirins: a hint into activity behavior.

Aspirin (acetylsalicylic acid, ASA) is the most popular non-steroidal anti-inflammatory drug. However, due to its action on cyclooxygenase and its acid nature, aspirin is associated with adverse gastrointestinal effects. In an effort to minimize these side effects, NO-donor and H2S-donor ASA co-drugs have been designed and tested. Their mass spectrometric behavior is now analyzed and reported. Positive ions were obtained by electrospray ionization involving protonation or alkali metal attachment. Their dissociation processes have been studied by collision induced dissociation in a triple quadrupole instrument. High mass accuracy measurements have been recorded on a Fourier transform ion cyclotron resonance mass spectrometer. The protonated molecules dissociate by an exclusive or largely prevailing path leading to acetyloxy-substituted benzoyl cation, namely an ASA unit. The process is reminiscent of the enzymatic hydrolysis, releasing intact ASA to a large extent. Only at higher collision energy does the formal ketene loss disrupt the ASA moiety. The gas phase chemistry of protonated ASA-releasing drugs develops along elementary dissociation steps analogous to the reactive processes in complex biological environments. This notion may provide a tool for preliminary testing of new compounds.

Identification and typing of Francisella tularensis with a highly automated genotyping assay

A PCR assay was developed to genotypically characterize Francisella tularensis and F. novicida. An integrated and partially redundant set of markers was selected to provide positive identification of these species, identify subspecies of F. tularensis and genotype 14 variable number tandem repeat (VNTR) markers. Assay performance was evaluated with 117 Francisella samples. Sample DNA was amplified, and the masses of the PCR products were determined with electrospray ionization/time of flight mass spectrometry (ESI-MS). The base compositions of the PCR amplicons were derived from these high-accuracy mass measurements and contrasted with databased information associated with each of the 25 assay markers. Species and subspecies determinations for all samples were fully concordant with results from established typing methods, and VNTR markers provided additional discrimination among samples. Sequence variants were observed with a number of assay markers, but these did not interfere with sample characterization, and served to increase the genetic diversity detected by the assay.

Investigation of the metabolism of monepantel in ovine hepatocytes by UHPLC/MS/MS

Monepantel (MOP) belongs to a new class of anthelmintic drugs known as aminoacetonitrile derivatives. It was approved for use in veterinary practice in Czech Republic in 2011. So far, biotransformation and transport of MOP in target animals have been studied insufficiently, although the study of metabolic pathways of anthelmintics is very important for the efficacy of safety of therapy and evaluation of the risk of drug-drug interactions. The aim of this study was to identify MOP metabolites and to suggest the metabolic pathways of MOP in sheep. For this purpose, primary culture of ovine hepatocytes was used as a model in vitro system. After incubation, medium samples and homogenates of hepatocytes were extracted separately using solid-phase extraction. Analysis was performed using a hybrid quadrupole-time-of-flight analyzer with respect to high mass accuracy measurements in full scan and tandem mass spectra for the confirmation of an elemental composition. The obtained results revealed S-oxidation to sulfoxide and sulfone and arene hydroxylation as MOP phase I biotransformations. From phase II metabolites, MOP glucuronides, sulfates, and acetylcysteine conjugates were found. Based on the obtained results, a scheme of the metabolic pathway of MOP in sheep has been proposed.

Transformation of cocaine during water chlorination

The stability of cocaine and its two main human metabolites, benzoylecgonine and ecgonine methyl ester, in chlorine-containing waters has been investigated by direct injection of different reaction time aliquots in a liquid chromatograph (LC) coupled to a quadrupole-time-of-flight mass spectrometer (QTOF-MS). Factors potentially affecting cocaine degradation (the only compound showing a significant decrease in the preliminary study) were evaluated in detail by means of a Box-Behnken experimental design. Sample pH resulted to be the most important variable, increasing both the rate of chlorination-mediated reactions and the ester hydrolysis process. From these reactions, and due to the high mass accuracy measurements obtained with the QTOF system, four by-products could be positively identified: benzoylecgonine, norcocaine, norbenzoylecgonine and N-formylnorcocaine. Finally, their formation and cocaine degradation yields were assessed under chlorination experiments with two real surface water samples. In one of them, showing a low anthropogenic impact, benzoylecgonine and norcocaine were notably generated even after only 1 h of reaction, whereas at higher contact times also norbenzoylecgonine and N-formylnorcocaine could be determined with a lower yield. On the other hand, the second sample, with a higher organic matter content, consumed rapidly the chlorine, so that only benzoylecgonine was produced. These findings point out the convenience of monitoring the described transformation products, in addition to the precursor illicit drug, during drinking water production, taking into account that cocaine traces might be present in water catchments and particularly in areas with high population densities.

Single Cell Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Application of mass spectrometry imaging (MS imaging) analysis to single cells was so far restricted either by spatial resolution in the case of matrix-assisted laser desorption/ionization (MALDI) or by mass resolution/mass range in the case of secondary ion mass spectrometry (SIMS). In this study we demonstrate for the first time the combination of high spatial resolution (7 μm pixel), high mass accuracy (<3 ppm rms), and high mass resolution (R = 100,000 at m/z = 200) in the same MS imaging measurement of single cells. HeLa cells were grown directly on indium tin oxide (ITO) coated glass slides. A dedicated sample preparation protocol was developed including fixation with glutaraldehyde and matrix coating with a pneumatic spraying device. Mass spectrometry imaging measurements with 7 μm pixel size were performed with a high resolution atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) imaging source attached to an Exactive Orbitrap mass spectrometer. Selected ion images were generated with a bin width of Δm/z = ±0.005. Selected ion images and optical fluorescence images of HeLa cells showed excellent correlation. Examples demonstrate that a lower mass resolution and a lower spatial resolution would result in a significant loss of information. High mass accuracy measurements of better than 3 ppm (root-mean-square) under imaging conditions provide confident identification of imaged compounds. Numerous compounds including small metabolites such as adenine, guanine, and cholesterol as well as different lipid classes such as phosphatidylcholine, sphingomyelin, diglycerides, and triglycerides were detected and identified based on a mass spectrum acquired from an individual spot of 7 μm in diameter. These measurements provide molecularly specific images of larger metabolites (phospholipids) in native single cells. The developed method can be used for a wide range of detailed investigations of metabolic changes in single cells.

High Mass Accuracy Phosphopeptide Identification Using Tandem Mass Spectra

Phosphoproteomics is a powerful analytical platform for identification and quantification of phosphorylated peptides and assignment of phosphorylation sites. Bioinformatics tools to identify phosphorylated peptides from their tandem mass spectra and protein sequence databases are important part of phosphoproteomics. In this work, we discuss general informatics aspects of mass-spectrometry-based phosphoproteomics. Some of the specifics of phosphopeptide identifications stem from the labile nature of phosphor groups and expanded peptide search space. Allowing for modifications of Ser, Thr, and Tyr residues exponentially increases effective database size. High mass resolution and accuracy measurements of precursor mass-to-charge ratios help to restrict the search space of candidate peptide sequences. The higher-order fragmentations of neutral loss ions enhance the fragment ion mass spectra of phosphorylated peptides. We show an example of a phosphopeptide identification where accounting for fragmentation from neutral loss species improves the identification scores in a database search algorithm by 50%.

Tandem mass spectrometry of bilin tetrapyrroles by electrospray ionization and collision-induced dissociation.

Bilins are metabolic products of hosts and bacteria on porphyrins, and are markers of health state and human waste contamination. Although bilin tandem mass spectrometry reports exist, their fragmentation behavior as a function of structure has not been compared, nor has fragmentation been examined as a function of collision energy. The fragmentation of bilins generated by positive ion mode electrospray ionization is examined by collision-induced dissociation (CID). CID on a quadrupole ion trap and on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer as a function of collision energy is compared. Methyl esterification was used to deduce which product ions contain the inner pyrrole rings. FT-ICR high mass accuracy measurements were used to determine the formulas of the resultant product ions. The central carbon's bonding to the inner pyrrole rings influences fragmentation. Bilirubin is unique because fragmentation adjacent to the central methylene group between innermost rings predominates, and loss of a terminal pyrrole is observed only with helium collision gas. The other bilins lose the terminal pyrroles first; as CID energy is increased, additional fragmentation due to neutral losses of small molecules such as H(2)O, CO, CO(2), and methanol occurs. Based on these observations, fragmentation schemes for the bilins are proposed that are strongly dependent on the molecular structure and collision energy; only bilirubin fragmentation is influenced significantly by the collision gas used. This report should have value in identification of this class of molecules for biomarker detection.

An Improved Calibration Method for the Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resononance Analysis of 15N-Metabolically-Labeled Proteome Digests Using a Mass Difference Approach

High mass measurement accuracy of peptides in enzymatic digests is critical for confident protein identification and characterization in proteomics research. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) can provide low or sub-ppm mass accuracy and ultrahigh resolving power. While for ESI-FT-ICR-MS, the mass accuracy is generally 1 ppm or better, with matrix-assisted laser desorption/ionization (MALDI)-FT-ICR-MS, the mass errors can vary from sub-ppm with internal calibration to over 100 ppm with conventional external calibration. A novel calibration method for (15)N-metabolically labeled peptides from a batch digest of a proteome is described which corrects for space charge induced frequency shifts in FT-ICR spectra without using an internal calibrant. This strategy utilizes the information from the mass difference between the (14)N/(15)N peptide peak pairs to correct for space charge induced mass shifts after data collection. A procedure for performing the mass correction has been written into a computer program and has been successfully applied to high-performance liquid chromatography-MALDI-FT- ICR-MS measurement of (15)N-metabolic labeled proteomes. We have achieved an average measured mass error of 1.0 ppm and a standard deviation of 3.5 ppm for 900 peptides from 68 MALDI-FT-ICR mass spectra of the proteolytic digest of a proteome from Methanococcus maripaludis.

High resolution ultra high pressure liquid chromatography–time-of-flight mass spectrometry dereplication strategy for the metabolite profiling of Brazilian Lippia species

Plants belonging to the Lippia genus have been widely used in ethnobotany throughout South and Central America and in tropical Africa as foods, medicines, sweeteners and in beverage flavouring. Various taxonomic problems involving some genera from Verbenaceae, including Lippia, have been reported. In this study, the metabolite profiling of fifteen extracts of various organs of six Lippia species was performed and compared using UHPLC–PDA-TOF-MS. Fourteen phenolic compounds that were previously isolated from L. salviaefolia Cham. and L. lupulina Cham. were used as references. The annotation of the remaining LC peaks was based on concomitant online high mass accuracy measurements and subsequent molecular formula assignments following these different steps: (i) elimination of non-coherent putative molecular formulae by heuristic filtering, (ii) verification of the occurrence of remaining molecular formulae in databases, (iii) cross search with reported compounds in the Lippia genus, (iv) match with reported UV spectra, (v) estimation of the chromatographic retention behaviour based on the logP parameter of reference compounds. This strategy is generic and time-saving, avoids isolation/purification procedures, enables an efficient LC peak annotation of most of the studied compounds and is well adapted for plant chemotaxonomic studies. Within this study, the interconversion of four flavanone glucoside isomers was additionally highlighted by analytical HPLC isolation and immediate analysis using fast UHPLC gradients. Dereplication results and hierarchical data analysis demonstrated that L. salviaefolia, L. balansae, L. velutina and L. sidoides displayed significant chemical similarities, while the compositions of L. lasiocalicyna and L. lupulina differed substantially.

Transient Ru-methyl formate intermediates generated with bifunctional transfer hydrogenation catalysts

Desorption electrospray ionization (DESI) coupled to high-resolution Orbitrap mass spectrometry (MS) was used to study the reactivity of a (β-amino alcohol)(arene)RuCl transfer hydrogenation catalytic precursor in methanol (CH(3)OH). By placing [(p-cymene)RuCl(2)](2) on a surface and spraying a solution of β-amino alcohol in methanol, two unique transient intermediates having lifetimes in the submillisecond to millisecond range were detected. These intermediates were identified as Ru (II) and Ru (IV) complexes incorporating methyl formate (HCOOCH(3)). The Ru (IV) intermediate is not observed when the DESI spray solution is sparged with Ar gas, indicating that O(2) dissolved in the solvent is necessary for oxidizing Ru (II) to Ru (IV). These proposed intermediates are supported by high-resolution and high mass accuracy measurements and by comparing experimental to calculated isotope profiles. Additionally, analyzing the bulk reaction mixture using gas chromatography-MS and nuclear magnetic resonance spectroscopy confirms the formation of HCOOCH(3). These results represent an example that species generated from the (β-amino alcohol)(arene)RuCl (II) catalytic precursor can selectively oxidize CH(3)OH to HCOOCH(3). This observation leads us to propose a pathway that can compete with the hydrogen transfer catalytic cycle. Although bifunctional hydrogen transfer with Ru catalysts has been well-studied, the ability of DESI to intercept intermediates formed in the first few milliseconds of a chemical reaction allowed identification of previously unrecognized intermediates and reaction pathways in this catalytic system.

C60 Secondary Ion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Secondary ion mass spectrometry (SIMS) has seen increased application for high spatial resolution chemical imaging of complex biological surfaces. The advent and commercial availability of cluster and polyatomic primary ion sources (e.g., Au and Bi cluster and buckminsterfullerene (C(60))) provide improved secondary ion yield and decreased fragmentation of surface species, thus improving accessibility of intact molecular ions for SIMS analysis. However, full exploitation of the advantages of these new primary ion sources has been limited, due to the use of low mass resolution mass spectrometers without tandem MS to enable enhanced structural identification capabilities. Similarly, high mass resolution and high mass measurement accuracy would greatly improve the chemical specificity of SIMS. Here we combine, for the first time, the advantages of a C(60) primary ion source with the ultrahigh mass resolving power and high mass measurement accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Mass resolving power in excess of 100 000 (m/Δm(50%)) is demonstrated, with a root-mean-square mass measurement accuracy below 1 part-per-million. Imaging of mouse brain tissue at 40 μm pixel size is shown. Tandem mass spectrometry of ions from biological tissue is demonstrated and molecular formulas were assigned for fragment ion identification.

Identification of phase I and phase II metabolites of benfluron and dimefluron in rat urine using high‐performance liquid chromatography/tandem mass spectrometry

Biotransformation products of two potential antineoplastic agents, benfluron and dimefluron, are characterized using our integrated approach based on the combination of high-performance liquid chromatography (HPLC) separation of phase I and phase II metabolites followed by photodiode-array UV detection and electrospray ionization tandem mass spectrometry (MS/MS). High mass accuracy measurement allows confirmation of an elemental composition and metabolic reactions according to exact mass defects. The combination of different HPLC/MS/MS scans, such as reconstructed ion current chromatograms, constant neutral loss chromatograms or exact mass filtration, helps the unambiguous detection of low abundance metabolites. The arene oxidation, N-oxidation, N-demethylation, O-demethylation, carbonyl reduction, glucuronidation and sulfation are typical mechanisms of the metabolite formation. The interpretation of their tandem mass spectra enables the distinction of demethylation position (N- vs. O-) as well as to differentiate N-oxidation from arene oxidation for both phase I and phase II metabolites. Two metabolic pathways are rather unusual for rat samples, i.e., glucosylation and double glucuronidation. The formation of metabolites that lead to a significant change in the chromophoric system of studied compounds, such as the reduction of carbonyl group in 7H-benzo[c]fluorene-7-one chromophore, is reflected in their UV spectra, which provides valuable complementary information to MS/MS data.

Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.

New mass data for the rp-process above Z = 32

High-accuracy mass measurements have been performed with the Penning trap mass spectrometer ISOLTRAP at ISOLDE/CERN. The short-lived nuclides 70, 71, 72, 73Se , 72, 73, 74, 75Br , and 98, 99, 100, 101, 103Ag have been measured with an average uncertainty of a few keV. The data are important input for nucleosynthesis calculations of the rp-process beyond Z = 32 .