Search

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.

Since sequencing of the human genome was completed, more than 500 genes have been annotated as proteases. Exploring the physiological role of each protease requires the identification of their natural substrates. However, the endogenous substrates of many of the human proteases are as yet unknown. Here we describe a new assay that addresses this problem. The assay, which easily can be automated, is based on the incubation of immobilized protein fractions, which may contain the natural substrate, with a defined protease. After concentrating the proteolytically released peptides by reversed-phase chromatography they are analyzed by tandem mass spectrometry and the substrates identified by database searching. The proof of principle in this study is demonstrated by incubating immobilized human plasma proteins with thrombin and by identifying by tandem mass spectrometry the fibrinopeptides, released by the action of thrombin from their natural substrate fibrinogen, in the reaction mixture.

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.

Fulvic acids are one of the largest classes of dissolved organic matter, but they are poorly defined and of unclear origin. Three fulvic acid isolates of different origin were analyzed by size-exclusion chromatography coupled to electrospray ionization-Fourier-transform ion cyclotron resonance (FTICR)-mass spectrometry, and molecular formulas for 700-1900 species in these isolates were derived. All three isolates show the same pattern in the elemental composition of their molecules and a large congruence in the molecular sets. It is proposed that the elemental and structural regularity of fulvic acid molecules does not indicate one common precursor material and formation process, but that this regularity is due to both the strong reworking of source materials in the environment and the valency of the three elements (C, H, O) from which most fulvic acid molecules are formed. Potential molecular formulas of fulvic acids were predicted for a mass range of 60 amu based on a few presumptions. A good agreement was found between the predicted and the detected molecular formulas, and it is concluded that (poly-)carboxylic acids with very limited number of hydroxy groups are the major compound class in fulvic acid isolates. It appears that fulvic acids are metastable molecules that characterize a state of transition of diverse precursor compounds during their oxidation.

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.

A dependable and stable CZE/ESI-MS interface has been constructed. To avoid instabilities in both, the capillary electrophoretic separation and the electrospray, the second of the three concentric capillaries in the three-layered sprayer has been replaced by an aluminum-coated fused-silica capillary with an inner diameter only slightly greater than the outer diameter of the separation capillary. By this means, the otherwise often observed destruction of the separation capillary ("electrodrilling") can be avoided completely due to the suppression of electrochemical processes leading to gas bubble formation at the tip of the sprayer. With some examples taken from different biochemical areas and by separation of natural compounds, the capability and the reliability of the modified sprayer as the central part of the interface are demonstrated.