Matrix-assisted Laser Desorption/ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Research Articles

Robust High-Throughput Proteomics Identification and Deamidation Quantitation of Extinct Species up to Pleistocene with Ultrahigh-Resolution MALDI-FTICR Mass Spectrometry.

Peptide mass fingerprinting (PMF) using MALDI-TOF mass spectrometry allows the identification of bone species based on their type I collagen sequence. In the archaeological or paleontological field, PMF is known as zooarchaeology mass spectrometry (ZooMS) and is widely implemented to find markers for most species, including the extinct ones. In addition to the identification of bone species, ZooMS enables dating estimation by measuring the deamidation value of specific peptides. Herein, we report several enhancements to the classical ZooMS technique, which reduces to 10-fold the required bone sample amount (down to the milligram scale) and achieves robust deamidation value calculation in a high-throughput manner. These improvements rely on a 96-well plate samples preparation, a careful optimization of collagen extraction and digestion to avoid spurious post-translational modification production, and PMF at high resolution using matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance (MALDI-FTICR) analysis. This method was applied to the identification of a hundred bones of herbivores from the Middle Paleolithic site of Caours (Somme, France) well dated from the Eemian Last Interglacial climatic optimum. The method gave reliable species identification to bones already identified by their osteomorphology, as well as to more challenging samples consisting of small or burned bone fragments. Deamidation values of bones originating from the same geological layers have a low standard deviation. The method can be applied to archaeological bone remains and offers a robust capacity to identify traditionally unidentifiable bone fragments, thus increasing the number of identified specimens and providing invaluable information in specific contexts.

In vivo microcapillary sampling coupled with matrix-assisted laser desorption/ionization fourier transform ion cyclotron resonance mass spectrometry for real-time monitoring of paraquat and diquat in living vegetables

An in vivo microcapillary sampling (MCS) method coupled with matrix-assisted laser desorption/ionization fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) analysis was utilized to monitor the real-time bipyridine quaternary ammonium herbicides concentrations and assess their uptake and elimination behaviors in living cabbage plants noninvasively. Under optimized conditions, the proposed method for paraquat (PQ) and diquat (DQ) determination showed wide linear ranges (7.81–500 μg/kg), low limits of detection (0.1–0.9 μg/kg), and good reproducibility. In vivo tracking results demonstrated that different absorption behaviors between PQ and DQ existed in living vegetables and DQ was more easily absorbed. Through decay kinetics model fitting, herbicide half-lives were 1.32 and 1.86 days for PQ and DQ, respectively. To summarize, in vivo MCS method provides valuable information on herbicide risks for agricultural production, which is suitable for temporal, spatial, and longitudinal studies in the same living system and multicompartmental studies in the same organism.

Improved N- and C-Terminal Sequencing of Proteins by Combining Positive and Negative Ion MALDI In-Source Decay Mass Spectrometry.

The development of various ionization and fragmentation techniques has been of key importance for establishing mass spectrometry (MS) as a powerful tool for protein characterization. One example of this is matrix-assisted laser desorption/ionization (MALDI) combined with in-source decay (ISD) fragmentation that allows mapping of N- and C-terminal regions of large proteins without the need for proteolysis. Positive ion mode ISD fragments are commonly assigned in the mass region above m/z 1000, while MALDI matrix ions generally hamper the detection of smaller singly charged fragments. The ultrahigh resolving power provided by Fourier transform ion cyclotron resonance (FT-ICR) MS partially overcomes this limitation, but to further increase the detection of smaller fragments we have revisited the application of negative ion mode MALDI-ISD and found good coverage of the peptide chain termini starting from c′2 and z′2 fragment ions. For the first time, we demonstrate that the combination of negative and positive ion MALDI FT-ICR MS is a useful tool to improve the characterization of mAbs. The different specificities of the two ion modes allowed us to selectively cover the sequence of the light and heavy chains of mAbs at increased sensitivity. A comprehensive evaluation of positive and negative ion mode MALDI-ISD FT-ICR MS in the m/z range 46–13 500 showed an increased sequence coverage for three standard proteins, namely, myoglobin, SiLuLite mAb, and NIST mAb. The data obtained in the two ion modes were, in part, complementary.

Localization of mercury and gold in cassava (Manihot esculenta Crantz).

The potential of cassava (Manihot esculenta Crantz.) for simultaneous Hg and Au phytoextraction was explored by investigating Hg and Au localization in cassava roots through Micro-Proton Induced X-Ray Emission, High-Resolution Transmission Electron Microscopy (HR-TEM) and X-Ray Diffractometry (XRD). The effect of Hg and Au in the cyanogenic glucoside linamarin distribution was also investigated using Matrix Assisted Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (MALDI-FT-ICR-MS) imaging. Hg was located mainly in the root vascular bundle of plants grown in 50 or 100μmolL-1 Hg solutions. Au was localized in the epidermis and cortex or in the epidermis and endodermis for 50 and 100μmolL-1 Au solutions, respectively. For 50μmolL-1 solutions of both Hg and Au, the two metals were co-localized in the epidermis. When the Hg concentrations were increased to 100μmolL-1, Au was still localized to a considerable extent in the epidermis while Hg was located in all root parts. HR-TEM and XRD revealed that Au nanoparticles were formed in cassava roots. MALDI-FT-ICR-MS imaging showed linamarin distribution in the roots of control and plants and metal-exposed plants thus suggesting that linamarin might be involved in Hg and Au uptake and distribution.

The Profile of Post-translational Modifications of Histone H1 in Chromatin of Mouse Embryonic Stem Cells.

Linker histone H1 is one of the main chromatin proteins which plays an important role in organizing eukaryotic DNA into a compact structure. There is data indicating that cell type-specific post-translational modifications of H1 modulate chromatin activity. Here, we compared histone H1 variants from NIH/3T3, mouse embryonic fibroblasts (MEFs), and mouse embryonic stem (ES) cells using matrix-assisted laser desorption/ ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS). We found significant differences in the nature and positions of the post-translational modifications (PTMs) of H1.3-H1.5 variants in ES cells compared to differentiated cells. For instance, methylation of K75 in the H1.2-1.4 variants; methylation of K108, K148, K151, K152 K154, K155, K160, K161, K179, and K185 in H1.1, as well as of K168 in H1.2; phosphorylation of S129, T146, T149, S159, S163, and S180 in H1.1, T180 in H1.2, and T155 in H1.3 were identified exclusively in ES cells. The H1.0 and H1.2 variants in ES cells were characterized by an enhanced acetylation and overall reduced expression levels. Most of the acetylation sites of the H1.0 and H1.2 variants from ES cells were located within their C-terminal tails known to be involved in the stabilization of the condensed chromatin. These data may be used for further studies aimed at analyzing the functional role played by the revealed histone H1 PTMs in the self-renewal and differentiation of pluripotent stem cells.

Rapid Characterization of Triacylglycerols in Edible Oils by Matrix-assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Abstract Composition and types of triacylglycerols (TAGs) in edible oils are closely related to our health. It is of vital significance to develop fast and high-efficiency methods for determination of TAGs. In this manuscript, qualitative analysis of TAGs was demonstrated using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). 2,5-Dihydroxybenzoic acid (DHB) was employed as matrix and dichloromethane was used as solvent for dissolving the edible oils. With laser power of 15%, laser frequency of 100 Hz, and 100 laser shots, relative standard deviation (RSD) less than 9% was obtained. Different kinds of edible oils could be directly distinguished from each other using MS and tandem MS (MS/MS) results. With confidence level of 95%, principal component analysis (PCA) results showed that 34 different kinds of edible oils were clearly classified. Moreover, down to 5% of doped canola in olive was identified directly, indicating that MALDI-FTICR-MS has the potential for rapid analysis and screening of edible oils.

Non-invasive and invasive diagnoses of aspergillosis in a rat model by mass spectrometry

Invasive pulmonary aspergillosis results in 450,000 deaths per year and complicates cancer chemotherapy, transplantations and the treatment of other immunosuppressed patients. Using a rat model of experimental aspergillosis, the fungal siderophores ferricrocin and triacetylfusarinine C were identified as markers of aspergillosis and quantified in urine, serum and lung tissues. Biomarkers were analyzed by matrix-assisted laser desorption ionization (MALDI) and electrospray ionization mass spectrometry using a 12T SolariX Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The limits of detection of the ferri-forms of triacetylfusarinine C and ferricrocin in the rat serum were 0.28 and 0.36 ng/mL, respectively. In the rat urine the respective limits of detection achieved 0.02 and 0.03 ng/mL. In the sera of infected animals, triacetylfusarinine C was not detected but ferricrocin concentration fluctuated in the 3–32 ng/mL range. Notably, the mean concentrations of triacetylfusarinine C and ferricrocin in the rat urine were 0.37 and 0.63 μg/mL, respectively. The MALDI FTICR mass spectrometry imaging illustrated the actual microbial ferricrocin distribution in the lung tissues and resolved the false-positive results obtained by the light microscopy and histological staining. Ferricrocin and triacetylfusarinine C detection in urine represents an innovative non-invasive indication of Aspergillus infection in a host.

MALDI-FT-ICR-MS for archaeological lipid residue analysis.

Soft-ionization methods are currently at the forefront of developing novel methods for analysing degraded archaeological organic residues. Here, we present little-used soft ionization method of matrix assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance-mass spectrometry (MALDI-FT-ICR-MS) for the identification of archaeological lipid residues. It is a high-resolution and sensitive method with low limits of detection capable of identifying lipid compounds in small concentrations, thus providing a highly potential new technique for the analysis of degraded lipid components. A thorough methodology development for analysing cooked and degraded food remains from ceramic vessels was carried out, and the most efficient sample preparation protocol is described. The identified components, also controlled by independent parallel analysis by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), demonstrate its capability of identifying very different food residues including dairy, adipose fats as well as lipids of aquatic origin. The results obtained from experimentally cooked and original archaeological samples prove the suitability of MALDI-FT-ICR-MS for analysing archaeological organic residues. Sample preparation protocol and identification of compounds provide future reference for analysing various aged and degraded lipid residues in different organic and mineral matrices.

Metabolomic profiling of prostate cancer by matrix assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry imaging using Matrix Coating Assisted by an Electric Field (MCAEF)

In this work, we combined the use of two MALDI matrices (quercetin and 9-aminoacridine), a recently developed new matrix coating technique – matrix coating assisted by an electric field (MCAEF), and matrix-assisted laser desorption/ionization – Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) to detect and image endogenous compounds in the cancerous and non-cancerous regions of three human prostate cancer (stage II) tissue specimens. After three rounds of imaging data acquisitions (i.e., quercetin for positive and negative ion detection and 9-aminoacridine for negative ion detection), and metabolite identification, a total of 1091 metabolites including 1032 lipids and 59 other metabolites were routinely detected and successfully localized. Of these compounds, 250 and 217 were only detected in either the cancerous or the non-cancerous regions respectively, although we cannot rule out the presence of these metabolites at concentrations below the detection limit. In addition, 152 of the other 624 metabolites showed differential distributions (p<0.05, t-test) between the two regions of the tissues. Further studies on a larger number of clinical specimens will need to be carried out to confirm this large number of apparently cancer-related metabolites. The successful determination of the spatial locations and abundances of these endogenous biomolecules indicated significant metabolism abnormalities – e.g., increased energy charge and under-expression of neutral acyl glycerides, in the prostate cancer samples. To our knowledge, this work has resulted in MALDI-MS imaging of the largest group of metabolites in prostate cancer thus far and demonstrated the importance of using complementary matrices for comprehensive metabolomic imaging by MALDI-MS. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Effect of ammonium perfluorooctanoate on acetylcholinesterase activity and inhibition using MALDI-FTICRMS

Ammonium perfluorooctanoate (APFO) is a commercially important compound, but its harm to people's health has raised widespread concern. In the past, the investigations into APFO and its degradation product (perfluorooctanoic acid, PFOA) were all about their effect on indicator compounds in animals and enzyme activities. Here, we provided a new suggestion to investigate the influence of APFO and PFOA. Acetylcholinesterase (AChE) was chosen as research subject to reflect the effect of external perfluorochemicals. We applied matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) to detect the activity of AChE rapidly and accurately. On this basis, not only AChE activity but also AChE inhibition was studied carefully. The presence of APFO and PFOA showed obvious increase of AChE activity. Moreover, addition of both APFO and PFOA had enhanced AChE inhibition from organophosphorous (OP) pesticide (irreversible inhibitor). Otherwise, the participation of APFO and PFOA had not increased AChE inhibitions from reversible inhibitor galantamine. These results might provide new insights into the effect of APFO and encourage the deep understanding about effect of APFO on human being.

MALDI-FTICR MS Imaging as a Powerful Tool to Identify Paenibacillus Antibiotics Involved in the Inhibition of Plant Pathogens

Nowadays, microorganisms are more and more often used as biocontrol agents for crop protection against diseases. Among them, bacteria of Bacillus and Paenibacillus genders are already used as commercial biocontrol agents. Their mode of action is supposed to be related to their production of antibiotics, such as cyclic lipopeptides, which exhibit great antimicrobial activities. We chose to work with a Paenibacillus polymyxa strain (Pp56) very resistant to various microorganisms. The bacteria were grown simultaneously with Fusarium oxysporum and we applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry to identify the antibiotics compounds present in the fungus growth inhibition area. We, therefore, identified fusaricidins A, B, and C and numerous members of the LI-F antibiotics family. MALDI-FTICR mass spectrometry imaging was then used to follow the diffusion of lipopeptides involved in the inhibitory activity over time. We analyzed the molecular content of the inhibitory area at different Pp56 and Fusarium incubation durations and concluded that some lipopeptides such as fusaricidin B and a mixture of LI-F05b/06b/08a were mainly involved in the defense mechanism of Pp56. Our study confirms that MALDI imaging may be a powerful tool to quickly determine which molecular species is involved in an antagonism with another microorganism, avoiding time-consuming steps of extraction, purification, and activity tests, which are still commonly used in microbiology.

Identification of reducing and nonreducing neutral carbohydrates by laser-enhanced in-source decay (LEISD) MALDI MS

In this work, laser-enhanced in-source decay (LEISD) technique of matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) was used to distinguish reducing and nonreducing carbohydrates. Interestingly, easier cleavage of (1 → 2)-linked glycosidic bonds for nonreducing carbohydrates containing D-fructofuranosyl units was observed in MALDI-FT-ICR-MS, which was in agreement with the result of theoretical calculation by the software package Gaussian 09. Importantly, no cross-ring cleavage of fructofuranosyl residues was detected in the LEISD spectra of nonreducing carbohydrates. LEISD method therefore offers an attractive alternative for fast and efficient differentiation of reducing and nonreducing carbohydrates, and the positions of nonreducing monosaccharide residues in a carbohydrate chain could be easily speculated.

Characterization of heme ions using MALDI-TOF MS and MALDI FT-ICR MS

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) and MALDI 15-T Fourier transform-ion cyclotron resonance (FT-ICR) MS were used to investigate the heme b ion from myoglobin and the heme c ion from tryptically digested cytochrome c. When the ion scan mode was changed from linear mode to reflectron mode in MALDI-TOF MS, the ratio of isotope distributions (intensities of M over M+1, where M is the integer m/z value of the most abundant ion which is 616 for heme b or 617 for heme c) changed for both heme ions. Similarly, when the ion scan mode was changed from a resolution of 8000 to 800,000 in MALDI 15-T FT-ICR MS, the same phenomenon was observed. The discrepancy in the isotopic distribution of heme ions in the MALDI 15-T FT-ICR MS analysis between the two resolutions was explained by the deconvolution process of overlapping peaks, while the difference in the isotopic distribution between the linear and reflectron modes in the MALDI-TOF MS analysis was explained by the presence of metastable ions which were only detectable in the linear mode of TOF analysis.

Determination of Small Molecular Aldehydes in Cigarette Smoke by Extraction and Derivatization in Single Drop

Extraction and derivatization in single drop (EDSD) coupled to matrix-assisted laser desorp- tion/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) was utilized to determine small molecular aldehydes in single puff smoke. Through employing Girard's reagent T as derivatization reagent, small molecular aldehydes (SMAs) transformed into quaternary ammonium salt, and the sensitivity of analysis for SMAs was significantly improved. Using this method, acetaldehyde and acrolein in single puff smoke were easily determined. This approach with an automatic potential provided a simple, rapid, and accurate procedure for the determination of SMAs in smoke.

Evaluation of combined matrix‐assisted laser desorption/ionization time‐of‐flight and matrix‐assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry experiments for peptide mass fingerprinting analysis

Peptide Mass Fingerprinting (PMF) is still of significant interest in proteomics because it allows a large number of complex samples to be rapidly screened and characterized. The main part of post-translational modifications is generally preserved. In some specific cases, PMF suffers from ambiguous or unsuccessful identification. In order to improve its reliability, a combined approach using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) was evaluated. The study was carried out on bovine serum albumin (BSA) digest. The influence of several important parameters (the matrix, the sample preparation method, the amount of the analyte) on the MOWSE score and the protein sequence coverage were evaluated to allow the identification of specific effects. A careful investigation of the sequence coverage obtained by each kind of experiment ensured the detection of specific peptides for each experimental condition. Results highlighted that DHB-FTICRMS and DHB- or CHCA-TOFMS are the most suited combinations of experimental conditions to achieve PMF analysis. The association (convolution) of the data obtained by each of these techniques ensured a significant increase in the MOWSE score and the protein sequence coverage.

Immunoglobulin G Glycopeptide Profiling by Matrix-Assisted Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation is essential for Fc-receptor-mediated activities. Changes in IgG Fc glycosylation have been found to be associated with various diseases. Here we describe a high-throughput IgG glycosylation profiling method. Sample preparation is performed in 96-well plate format: IgGs are purified from 2 microL of human plasma using immobilized protein A. IgGs are cleaved with trypsin, and the resulting glycopeptides are purified by reversed-phase or hydrophilic interaction solid-phase extraction. Glycopeptides are analyzed by intermediate pressure matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Notably, both dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) matrixes allowed the registration of sialylated as well as nonsialylated glycopeptides. Data were automatically processed, and IgG isotype-specific Fc glycosylation profiles were obtained. The entire method showed an interday variation below 10% for the six major glycoforms of both IgG1 and IgG2. The method was found suitable for isotype-specific high-throughput IgG glycosylation profiling from human plasma. As an example we successfully applied the method to profile the IgG glycosylation of 62 human samples.

The Effect of Preanalytical Factors on Stability of the Proteome and Selected Metabolites in Cerebrospinal Fluid (CSF)

To standardize the use of cerebrospinal fluid (CSF) for biomarker research, a set of stability studies have been performed on porcine samples to investigate the influence of common sample handling procedures on proteins, peptides, metabolites and free amino acids. This study focuses at the effect on proteins and peptides, analyzed by applying label-free quantitation using microfluidics nanoscale liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (chipLC-MS) as well as matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) and Orbitrap LC-MS/MS to trypsin-digested CSF samples. The factors assessed were a 30 or 120 min time delay at room temperature before storage at -80 degrees C after the collection of CSF in order to mimic potential delays in the clinic (delayed storage), storage at 4 degrees C after trypsin digestion to mimic the time that samples remain in the cooled autosampler of the analyzer, and repeated freeze-thaw cycles to mimic storage and handling procedures in the laboratory. The delayed storage factor was also analyzed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for changes of metabolites and free amino acids, respectively. Our results show that repeated freeze/thawing introduced changes in transthyretin peptide levels. The trypsin digested samples left at 4 degrees C in the autosampler showed a time-dependent decrease of peak areas for peptides from prostaglandin D-synthase and serotransferrin. Delayed storage of CSF led to changes in prostaglandin D-synthase derived peptides as well as to increased levels of certain amino acids and metabolites. The changes of metabolites, amino acids and proteins in the delayed storage study appear to be related to remaining white blood cells. Our recommendations are to centrifuge CSF samples immediately after collection to remove white blood cells, aliquot, and then snap-freeze the supernatant in liquid nitrogen for storage at -80 degrees C. Preferably samples should not be left in the autosampler for more than 24 h and freeze/thaw cycles should be avoided if at all possible.

Extraction and derivatization in single drop coupled to MALDI-FTICR-MS for selective determination of small molecule aldehydes in single puff smoke

Extraction and derivatization in single drop (EDSD) coupled to matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) was utilized to determine small molecular aldehydes (SMAs) in single puff smoke. A methanol solution of diphenylamine, 2,5-dihydroxybenzoic acid was used for extraction/derivatization of SMAs and a microdrop of the extraction solution containing the ionic derivatization products of SMAs could be directly deposited in the MALDI target to perform the measurement of SMAs in the cigarette smoke on puff level by MALDI-FTICR-MS. As a result, a consecutive operation of sample pre-treating and MALDI sample plate preparation was achieved. SMAs were derivatized by the reaction with diphenylamine and transformed a stable Schiff base bearing a quaternary ammonium group, and the sensitivity of analysis for SMAs was significantly improved. Good sample homogeneity in in-dot was achieved by adding diphenylamine into the solvent for EDSD and a satisfying repeatability (R.S.D. = 7.3% for formaldehyde, n = 5) of MALDI-FTICR-MS signals was obtained. This solvent- and reagent-minimized approach with an automatic potential provided a simple, rapid, and accurate procedure for the determination of SMAs in smoke.

Investigations with O‐linked protein glycosylations by matrix‐assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry

Posttranslational modifications such as glycosylation can play a fundamental role in signaling pathways that transform an ordinary cell into a malignant one. The development of a protocol to detect these changes in the preliminary stages of disease can lead to a sensitive and specific diagnostic for the early detection of malignancies such as ovarian cancer in which differential glycan patterns are linked to etiology and progression. Small variations in instrument parameters and sample preparation techniques are known to have significant influence on the outcome of an experiment. For an experiment to be effective and reproducible, these parameters must be optimized for the analyte(s) under study. We present a detailed examination of sample preparation and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) analysis of O-linked glycans globally cleaved from mucin glycoproteins. Experiments with stable isotope-labeled biomolecules allowed for the establishment of appropriate acquisition times and excitation voltages for MALDI-FT-ICR-MS of oligosaccharides. Quadrupole ion guide optimization studies with mucin glycans identified conditions for the comprehensive analysis of the entire mass range of O-linked carbohydrates in this glycoprotein. Separately optimized experimental parameters were integrated in a method that allowed for the effective study of O-linked glycans.

High resolution mass spectrometric brain proteomics by MALDI-FTICR-MS combined with determination of P, S, Cu, Zn and Fe by LA-ICP-MS

The combination of atomic and molecular mass spectrometric methods was applied for characterization and identification of several human proteins from Alzheimer's diseased brain. A brain protein mixture was separated by two-dimensional (2D) gel electrophoresis and the protein spots were fast screened by microlocal analysis using LA-ICP-MS (laser ablation inductively coupled plasma mass spectrometry) in respect to phosphorus, sulfur, copper, zinc and iron content. Five selected protein spots in 2D gel containing these elements were investigated after tryptic digestion by matrix assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Than element concentrations (P, Cu, Zn and Fe) were determined in three identified human brain proteins by LA-ICP-MS in the 2D gel. Results of structure analysis of human brain proteins by MALDI-FTICR-MS were combined with those of the direct determination of phosphorus, copper, zinc and iron concentrations in protein spots with LA-ICP-MS. From the results of atomic and molecular mass spectrometric techniques the human brain proteins were characterized in respect to their structure, sequence, phosphorylation state and metal content as well.