Abstract. The molecular composition of particle phase ozonolysis products of o•-pinene is investigated to comprehend the aerosol formation process following the VOC oxidation, focusing on an understanding of new particle formation. Two analytical approaches are applied to identify lowvolatile oxidation products in the particle phase; off-line investigations using preconcentration on Tenax TA © followed by solvent extraction and liquid chromatography/mass spectrometry as well as an on-line technique, in which the organic aerosols are introduced directly into the ion source of a mass spectrometer (atmospheric pressure chemical ionization / mass spectrometry (APCI/MS)). Both techniques showed the formation of difunctional carboxylic acids, compounds whose physico-chemical properties will govern most of their mass into the particle phase. Furthermore, stable binary diacid adducts could be identified by MSn-experiments. These observations might give insight into the process of new particle formation by heteromolecular homogeneous nucleation, indicating that the initial cluster formation cannot be described by macroscopic properties of single oxidation products. Instead, strong intermolecular forces between different diacids might play a key role in the formation of initial nuclei and their subsequent growth.

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures. Proteomics as a field of research is based on the characterization of an entire proteome of a biological system. A variety of approaches have been developed during the last decades to characterize such mixtures of proteins and peptides, and necessarily, all of them use separation techniques. At the protein level, separation has been achieved using 2-D 1 gel electrophoresis (1) and densitometry of stained proteins or fluorescence detection (2). After digestion of the proteins, peptides were identified using liquid chromatography, mass spectrometry, or both (3, 4). However, this information was only qualitative. It became rapidly evident that quantitative data were definitively required, e.g. for the characterization of dynamic biological systems or the search for biomarkers in clinical proteomics. Subsequently methods have been developed for the quantitative determination of proteins and peptides mainly based on chemical or metabolic isotopic labeling combined with LC/MS n detection (5, 6). Label-free LC/MS quantitative strategies are under development as well (7).Using such techniques, the investigation of changes of the proteome in biological systems has become possible. However, o...

A method for phosphopeptide identification by capillary liquid chromatography (muLC) interfaced alternatively to element mass spectrometry (inductively coupled plasma mass spectrometry, ICPMS) and to electrospray ionization mass spectrometry (ESI-MS) is described. ICPMS is used for 31P detection and ESI-MS provides the corresponding molecular weight information. Alignment of the two separate muLC runs is performed using the baseline distortion at the elution front, which shows up in both muLC-ICPMS and muLC-ESI-MS. Both a quadrupole and a magnetic sector field mass analyzer were used in combination with ICP. The detection limit achieved for the muLC-ICP-HRMS runs is approximately 0.1 pmol of phosphopeptide injected. Without any further precautions, contamination by phosphate-containing compounds at this level was found to be uncritical. The method is demonstrated for the analysis of a complex mixture of synthetic phosphopeptides and a set of tryptic digests of three phosphoproteins. These include beta-casein, activated human MAP kinase ERK1, and protein kinase A catalytic subunit. The tryptic phosphopeptides of these proteins could all be detected and identified by our new strategy. Analysis of three fractions of protein kinase A catalytic subunit with different phosphorylation status gives direct access to the order in which the phosphorylation of the four phosphorylation sites occurs. The two most important aspects of using muLC-ICPMS with 31P detection for phosphopeptide identification are (i) that a high selectivity is achieved and (ii) that the signal intensity is independent of the chemical form of phosphorus and directly proportional to the molar amount of 31P in the muLC eluate. Thus, muLC-ICPMS with 31P detection is introduced as a new, robust, and specific method in phosphoproteomics.

The water-soluble organic fractions of aerosol samples collected in Riverside, CA, in summer 2005 were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Elemental compositions of about 1000 molecular species were determined in the range m/z 220-420, and four series of organic compounds were identified, fulvic acids, and S-containing, N-containing, and S- and N-containing molecules. Low-resolution product ion spectra proved the presence of organosulfates, organonitrates, and mixed organosulfates and -nitrates that appear to be structurally closely related to each other and to the fulvic acids. This is the first unambiguous detection of fulvic acid molecules and sulfated components in atmospheric aerosol and the first detection even of nitrated analogues. These species provide new clues to the nature of particulate organic matter in atmospheric aerosol.

What is already known about this subject • In recent years, it has been suggested that hair follicles represent important shunt routes into the skin for drugs and chemicals [1–3]. • In vitro studies have shown the importance of skin appendages for skin penetration by hydrophilic compounds [4]. Investigation of follicular penetration in vivo has been difficult due to the absence of appropriate analytical methods or suitable animal model systems. • Recently, a new method was described that quantifies follicular penetration in vivo by using selective closure of hair follicles [5]. • Caffeine is frequently used in skin penetration experiments as a model for highly water‐soluble compounds. Occlusion [6] and skin thickness [7] seem to have little influence on the penetration of caffeine. However, percutaneous absorption rates for caffeine exhibit regional skin differences in humans in vivo[1]. What this study adds • The results of the present study demonstrate that a fast drug delivery of caffeine occurs through shunt routes. Therefore, hair follicles are considerable weak spots in our protective sheath against penetration into the body by hydrophilic substances. • We showed that there is a quantitative distinction between follicular penetration and interfollicular diffusion of caffeine in vivo. • These findings are of importance for the development and optimization of topically applied drugs and cosmetics. In addition, such properties must be considered in the development of skin protection measures. Aims The skin and its appendages are our protective shield against the environment and are necessary for the maintenance of homeostasis. Hypotheses concerning the penetration of substances into the skin have assumed diffusion through the lipid domains of the stratum corneum. It is believed that while hair follicles represent a weakness in the shield, they play a subordinate role in the percutaneous penetration processes. Previous investigation of follicular penetration has mostly addressed methodical and technical problems. Our study utilized a selective closure technique of hair follicle orifices in vivo, for the comparison of interfollicular and follicular absorption rates of caffeine in humans. Methods Every single hair follicle within a delimited area of skin was blocked with a microdrop of a special varnish‐wax‐mixture in vivo. Caffeine in solution was topically applied and transcutaneous absorption into the blood was measured by a new surface ionization mass spectrometry (SI/MS) technique, which enabled a clear distinction to be made between interfollicular and follicular penetration of a topically applied substance. Results Caffeine (3.75 ng ml−1) was detected in blood samples, 5 min after topical application, when the follicles remained open. When the follicles were blocked, caffeine was detectable after 20 min (2.45 ng ml−1). Highest values (11.75 ng caffeine ml−1) were found 1 h after application when the follicles were open. Conclusions Our findings demonstrate that hair follicles are considerable weak spot...

Dissolved organic matter isolated from the deep Atlantic Ocean and fractionated into a so-called hydrophobic (HPO) fraction and a very hydrophilic (HPI) fraction was analyzed for the first time by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) to resolve the molecular species, to determine their exact masses, and to calculate their molecular formulas. The elemental composition of about 300 molecules was identified. Those in the HPO fraction (14C age of 5100 year) are very similar to much younger freshwater fulvic acids, but less aromatic and more oxygenated molecules are more frequent. This trend continues toward the HPI fraction and may indicate biotic and abiotic aging processes that this material experienced since its primary production thousands of years ago. In the HPI fraction series of nitrogenous molecules containing one, two, or three nitrogens were identified by FTICR-MS. Production spectra of the nitrogenous molecules suggest that the nitrogen atoms in these molecules are included in the (alicyclic) backbone of these molecules, possibly in reduced form. These mass spectrometric data suggest that a large set of stable fulvic acids is ubiquitous in all aquatic compartments. Although sources may differ, their actual composition and structure appears to be quite similar and largely independent from their source, because they are the remainder of intensive oxidative degradation processes.

We have developed lanthanide labeling strategies for antibodies to adapt conventional biochemical workflows like Western blot immunoassays for detection by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis with a special interest to apply the multi-element capabilities of ICP-MS for the design of multiplexed immunoassays. In this paper the lanthanide labeling of antibodies with MeCAT was investigated and the reaction conditions were optimized for application in a Western blot immunoassay analyzed by LA-ICP-MS. Furthermore, the MeCAT labeling strategy was compared with two other commercially available labeling reagents, MAXPARÔ and SCN-DOTA. As a proof-of-principle experiment chemically induced alterations of cytochrome P450 protein expression were investigated and the suitability of the differentially labeled antibodies for Western blot immunoassays of a complex liver microsomal protein fraction was tested. Limits of detection (LODs) in the lower fmol range were reached in the Western blot application using MeCAT and MAXPARÔ as element labeling reagents, whereas even sub-fmol LODs can be achieved in a dot blot experiment for the pure antibodies.

Metabolites, lipids, and other small molecules are key constituents of tissues supporting cellular programs in health and disease. Here, we present METASPACE, a community-populated knowledge base of spatial metabolomes from imaging mass spectrometry data. METASPACE is enabled by a high-performance engine for metabolite annotation in a confidence-controlled way that makes results comparable between experiments and laboratories. By sharing their results publicly, engine users continuously populate a knowledge base of annotated spatial metabolomes in tissues currently including over 3000 datasets from human cancer cohorts, whole-body sections of animal models, and various organs. The spatial metabolomes can be visualized, explored and shared using a web app as well as accessed programmatically for large-scale analysis. By using novel computational methods inspired by natural language processing, we illustrate that METASPACE provides molecular coverage beyond the capacity of any individual laboratory and opens avenues towards comprehensive metabolite atlases on the levels of tissues and organs.