| 1. |
- Kjellström, Sven
(författare)
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Sampling and Selective Detection of Peptides and Proteins Emphazing Microdialysis,Bio-recognition Assays and MALDI-TOF MS
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The use of microdialysis sampling and flow immunochemical detection as well as MALDI-TOF MS for peptide and protein analysis is described. Sampling of peptides and smaller proteins was investigated with different membranes. Cyclodextrines additives and an on-line push-pull microdialysis sampling method was used with the result of a higher sampling efficiency compared to conventional microdialysis sampling. Real-time flow immuno assays systems were developed for the quantification of Interleukin-8 and Interleukin10 in cell samples. Optimisation with regards to affinity support selection as well as carrier buffer composition were performed. Connection of capillary LC with MALDI-TOF MS was made utilising an m-dispenser technique enabling sample handling of picoliter volumes. A novel sample preparation technique for the uniform coating of MALDI targets was developed.
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| 2. |
- Miliotis, Tasso, et al.
(författare)
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Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry
- 2000
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 886:1-2, s. 99-110
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Tidskriftsartikel (refereegranskat)abstract
- An integrated protein microcharacterization/identification platform has been developed. The system has been designed to allow a high flexibility in order to tackle challenging analytical problems. The platform comprises a cooled microautosampler, an integrated system for microcolumn HPLC, and a capillary reversed-phase column that is interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) system via a low internal volume flow-through microdispenser. The chromatographic separation is continuously transferred onto a MALDI target plate as discrete spots as the dispenser ejects bursts of droplets of the column effluent in a precise array pattern. A refrigerated microfraction collector was coupled to the outlet of the flow-through microdispenser enabling enrichment and re-analysis of interesting fractions. The use of target plates pre-coated with matrix simplified and increased the robustness of the system. By including a separation step prior to the MALDI-TOF-MS analysis and hereby minimizing suppression effects allowed us to obtain higher sequence coverage of proteins compared to conventional MALDI sample preparation methodology. Additionally, synthetic peptides corresponding to autophosphorylated forms of the tryptic fragment 485-496 (ALGADDSYYTAR) of tyrosine kinase ZAP-70 were identified at sensitivities reaching 150 amol. Copyright (C) 2000 Elsevier Science B.V.
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| 3. |
- Miliotis, Tasso, et al.
(författare)
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Ready-made matrix-assisted laser desorption/ionization target plates coated with thin matrix layer for automated sample deposition in high-density array format.
- 2002
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Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 1097-0231 .- 0951-4198. ; 16:2, s. 117-126
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Tidskriftsartikel (refereegranskat)abstract
- The methodology for ready-made matrix-assisted laser desorption/ionization (MALDI) target plates covered with an optimized thin layer consisting of matrix and nitrocellulose has been developed. Piezoelectric microdispensing enabled sample depositions in a high-density array format of 2000 sample depositions on a conventionally sized target plate (45 x 47 mm). The sample depositions were made reproducibly in a fully automated mode by using an in-house developed computer-controlled piezoelectric flow-through microdispenser. Additionally, the piezoelectric technique facilitated significant analyte enrichment that increased the detection sensitivity. The MS signal was obtained rapidly, generally within ten laser pulses. An airbrush device was used to generate a fine spray of matrix and nitrocellulose dissolved in acetone. The acetone evaporated instantly when reaching the target plate leaving the entire surface with a thin and uniform matrix/nitrocellulose coating consisting of very small crystals of matrix embedded in the nitrocellulose. These crystals acted as a seed-layer on subsequent analyte depositions, rendering homogeneous sample spots when using alpha-cyano-4-hydroxycinnamic acid (CHCA) as matrix. The relative standard deviation of the signal intensity between spots was (20-30)% (n = 30). The detection sensitivity was improved by restricting the sample spot diameter to 300 microm. The spot size was affected by the deposition rate and the evaporation rate of the dispensed sample volume. Mass spectra of a 25-amol peptide mixture deposition were successfully recorded.
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