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- Bergquist, Jonas, et al.
(författare)
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Peptide Mapping of Proteins in Human Body Fluids using Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
- 2002
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Ingår i: Mass spectrometry reviews (Print). - : Wiley. - 0277-7037 .- 1098-2787. ; 21:1, s. 2-15
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Tidskriftsartikel (refereegranskat)abstract
- Human body fluids have been rediscovered in the postgenomic era as great sources of biological markers and perhaps particularly as sources of potential protein biomarkers of disease. Analytical tools that allow rapid screening, low sample consumption, and accurate protein identification are of great importance in studies of complex biological samples and clinical diagnosis. Mass spectrometry is today one of the most important analytical tools with applications in a wide variety of fields. One of the fastest growing applications is in proteomics, or the study of protein expression in an organism. Mass spectrometry has been used to find post-translational modifications and to identify key functions of proteins in the human body. In this study, we review the use of human body fluids as sources for clinical markers and present new data that show the ability of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to identify, and characterize proteins in four human body fluids: plasma, cerebrospinal fluid (CSF), saliva, and urine. The body fluids were tryptically digested without any prior separation, purification, or selection, and the digest was introduced into a 9.4 T FTICR mass spectrometer by direct-infusion electrospray ionization (ESI). Even though these samples represent complex biological mixtures, the described method provides information that is comparable with traditional 2D-PAGE data. The sample consumption is extremely low, a few microliters, and the analysis time is only a few minutes. It is, however evident that the separation of proteins and/or peptides must be included in the methodology in order to detect low-abundance proteins and other proteins of biological relevance.
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| 4. |
- Palmblad, Magnus, et al.
(författare)
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Analysis of Enzymatically Digested Proteins and Protein Mixtures using a 9.4 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
- 2000
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Ingår i: Rapid Communications in Mass Spectrometry. - 0951-4198 .- 1097-0231. ; 14:12, s. 1029-1034
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Tidskriftsartikel (refereegranskat)abstract
- A commercially available 9.4 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was applied in the analysis of tryptic digests of protein mixtures without any separation. First, the method was demonstrated on a mixture of tryptic digests of equine cytochrome c, equine myoglobin and bovine serum albumin. The same method was then applied to human plasma from a healthy blood donor. Computer programs were employed to simplify analysis of the complex spectra. The 2745 peaks in the human plasma electrospray ionization FTICR spectrum could be reduced to 1165 isotopic clusters and 669 unique masses. Out of these, 82 masses matched tryptic fragments of serum albumin with mass measurement errors less than 10 ppm, covering 93% of the sequence. Another 16 masses were assigned to tryptic fragments of transferrin, covering 41% of the sequence on the 10 ppm mass measurement error level (14 within 2 ppm). The mass measurement errors were approximately normal distributed with a standard deviation of 1.7 ppm. This demonstrates the feasibility of combining the ultra-high mass resolving power and accuracy of FTICR mass spectrometry with automated computer analysis for investigating complex biological matrices.
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| 7. |
- Nilsson, Stefan, et al.
(författare)
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Explorative Study of the Protein Composition of Amniotic Fluid by Liquid Chromatography Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
- 2004
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). ; 3:4, s. 884-889
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Tidskriftsartikel (refereegranskat)abstract
- To explore the suitability of FTICR mass spectrometry for the analysis of the protein composition of amniotic fluid (AF), an AF sample from 15 weeks gestation from a healthy 36-year-old woman was tryptically digested, with or without prior serum albumin removal. The tryptic peptides were separated by gradient capillary liquid chromatography (LC) followed by electrospray ionization (ESI) and mass spectrometric detection with a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR). The obtained data underwent computer-aided mathematical and statistical evaluation to extract significant tryptic peptide patterns from human proteins. Forty-three proteins were putatively identified; among them were known protein constituents of amniotic fluid, but also many that not have been detected before. The removal of serum albumin prior to tryptic digestion reduced ion suppression from abundant HSA fragments. The protein analysis of amniotic fluid by albumin removal, tryptic digestion and LC/FT-ICR-MS analysis was found to be a straightforward technique.
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| 8. |
- Palmblad, Magnus, et al.
(författare)
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A 9.4 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer : Description and Performance
- 2000
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Ingår i: European journal of mass spectrometry. - 1469-0667 .- 1751-6838. ; 6:3, s. 267-275
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Tidskriftsartikel (refereegranskat)abstract
- 9.4 Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers (Bruker BioAPEX-94e) have been installed at the Division of Ion Physics, Uppsala University, and at the Department of Chemistry, University of Warwick, The BioAPEX-94e FT-ICR instrument is built around a high-field, superconducting magnet and a platform with easily interchangeable ion sources [matrix-assisted laser desorption/ionisation (MALDI), secondary ion mass spectrometry (SIMS), electrospray ionisation (ESI) and electron impact/chemical ionisation (EI/CI)I. In this paper a technical description of the instrument is given. Outstanding performance characteristics are demonstrated, notably clear resolution of C59N+ and (C58C2+)-C-13 (mass difference 3.65 mDa) and mass measurement accuracy at the low ppm level. A wide range of applications in Warwick and Uppsala is described, demonstrating the versatility and high performance of the instrument.
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| 9. |
- Palmblad, Magnus, et al.
(författare)
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Automatic Analysis of Hydrogen/Deuterium Exchange Mass Spectra of Peptides and Proteins using Calculations of Isotopic Distributions
- 2001
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Ingår i: Journal of the American Society for Mass Spectrometry. - 1044-0305 .- 1879-1123. ; 12:11, s. 1153-1162
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Tidskriftsartikel (refereegranskat)abstract
- High mass-resolving power has been shown to be useful for studying the conformational dynamics of proteins by hydrogen/deuterium (H/D) exchange. A computer algorithm was developed that automatically identifies peptides and their extent of deuterium incorporation from H/D exchange mass spectra of enzymatic digests or fragment ions produced by collisionally induced dissociation (CID) or electron capture dissociation (ECD). The computer algorithm compares measured and calculated isotopic distributions and uses a fast calculation of isotopic distributions using the fast Fourier transform (FFT). The algorithm facilitates rapid and automated analysis of H/D exchange mass spectra suitable for high-throughput approaches to the study of peptide and protein structures. The algorithm also makes the identification independent on comparisons with undeuterated control samples. The applicability of the algorithm was demonstrated on simulated isotopic distributions as well as on experimental data, such as Fourier transform ion cyclotron resonance (FTICR) mass spectra of myoglobin peptic digests, and CID and ECD spectra of substance P.
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| 10. |
- Palmblad, Magnus, 1973-
(författare)
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Identification and Characterization of Peptides and Proteins using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mass spectrometry has in recent years been established as the standard method for protein identification and characterization in proteomics with excellent intrinsic sensitivity and specificity. Fourier transform ion cyclotron resonance is the mass spectrometric technique that provides the highest resolving power and mass accuracy, increasing the amount of information that can be obtained from complex samples. This thesis concerns how useful information on proteins of interest can be extracted from mass spectrometric data on different levels of protein structure and how to obtain this data experimentally. It was shown that it is possible to analyze complex mixtures of protein tryptic digests by direct infusion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and identify abundant proteins by peptide mass fingerprinting. Coupling on-line methods such as liquid chromatography and capillary electrophoresis increased the number of proteins that could be identified in human body fluids. Protein identification was also improved by novel statistical methods utilizing prediction of chromatographic behavior and the non-randomness of enzymatic digestion. To identify proteins by short sequence tags, electron capture dissociation was implemented, improved and finally coupled on-line to liquid chromatography for the first time. The combined techniques can be used to sequence large proteins de novo or to localize and characterize any labile post-translational modification. New computer algorithms for the automated analysis of isotope exchange mass spectra were developed to facilitate the study of protein structural dynamics. The non-covalent interaction between HIV-inhibitory peptides and the oligomerization of amyloid β-peptides were investigated, reporting several new findings with possible relevance for development of anti-HIV drug therapies and understanding of fundamental mechanisms in Alzheimer’s disease.
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