Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry Sample Preparation Techniques Designed for Various Peptide and Protein Analytes

Kussmann, Martin; Nordhoff, Eckhard; Rahbek‐Nielsen, Henrik; Haebel, Sophie; Rossel‐Larsen, Martin; Jakobsen, Lene; Gobom, Johan; Mirgorodskaya, Ekatarina; Kroll‐Kristensen, Anne; Palm‖, Lisbeth; Roepstorff, Peter

doi:10.1002/(sici)1096-9888(199706)32:6<593::aid-jms511>3.3.co;2-4

Cited by 144 publications

(192 citation statements)

References 2 publications

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“…Even if all the eluate was to be collected on the same MALDI sample spot area a less dispersed sample plug will provide better control over the matrix/analyte ratio and the crystallization process which is critical for the MS results [7,8]. With respect to the sample throughput of the system the dispersion of any fluidic element will add process time to each step in a microextraction sequence; sample loading, washing and extraction.…”

Section: Discussionmentioning

confidence: 99%

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation

et al. 2002

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A recently introduced silicon microextraction chip (SMEC), used for on-line proteomic sample preparation, has proved to facilitate the process of protein identification by sample clean up and enrichment of peptides. It is demonstrated that a novel grid-SMEC design improves the operating characteristics for solid-phase microextraction, by reducing dispersion effects and thereby improving the sample preparation conditions. The structures investigated in this paper are treated both numerically and experimentally. The numerical approach is based on finite element analysis of the microfluidic flow in the microchip. The analysis is accomplished by use of the computational fluid dynamics-module FLOTRAN in the ANSYS software package. The modeling and analysis of the previously reported weir-SMEC design indicates some severe drawbacks, that can be reduced by changing the microextraction chip geometry to the grid-SMEC design. The overall analytical performance was thereby improved and also verified by experimental work. Matrix-assisted laser desorption/ionization mass spectra of model peptides extracted from both the weir-SMEC and the new grid-SMEC support the numerical analysis results. Further use of numerical modeling and analysis of the SMEC structures is also discussed and suggested in this work.

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Section: Discussionmentioning

confidence: 99%

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation

et al. 2002

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“…Custom-made chromatographic microcolumns were used for desalting and concentration of the peptide mixture prior to mass spectrometric analysis [16,18]. A column consisting of 100-300 nL of Poros RP R2 (R2) material (20 mm bead size; PerSeptive Biosystems, Framingham, MA, USA) or graphite powder (GP) (obtained from prepacked 1 mL graphite columns (Alltech, Sydney, Australia) or activated charcoal from Sigma (C-5510)) was packed in a constricted GELoader tip (Eppendorf, Hamburg, Germany).…”

Section: Desalting and Concentrationmentioning

confidence: 99%

“…Some of these problems have been overcome with the introduction of custommade disposable microcolumns, which have been used as a fast clean-up step prior to mass analysis [15]. This methodology was later simplified by using Eppendorf GELoader tips packed with chromatographic material [16][17][18] or commercially available prepacked RP microcolumns (e.g. ZipTips TM , Millipore, Bedford, MA, USA).…”

Section: Introductionmentioning

confidence: 99%

Graphite powder as an alternative or supplement to reversed-phase material for desalting and concentration of peptide mixtures prior to matrix-assisted laser desorption/ionization-mass spectrometry

2002

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The success attributed to identification and characterization of gel separated proteins by mass spectrometry (MS) is highly dependent on the percentage of an entire sequence covered by matching peptides derived from enzymatic digestion. Desalting and concentration of peptide mixtures on reversed-phase (RP) microcolumns prior to mass spectrometric analysis have resulted in increased signal-to-noise ratio and sensitivity, and consequently higher sequence coverage. A large proportion of peptides, however, remains undetected by MS presumably because they are lost during sample preparation on microcolumns, or are suppressed in the ionization process. We report here the use of graphite powder packed in constricted GELoader tips as an alternative to RP microcolumns for desalting and concentration of peptide mixtures prior to MS. Such columns are able to retain small and/or hydrophilic peptides that can be lost when using RP microcolumns. In addition, we show that samples contaminated with small biological polymers can readily be analyzed using graphite powder rather than RP microcolumns, since the polymer molecules bind strongly to graphite and are not eluted with the peptides.

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“…Alternatively, the resulting peptides were extracted by adding 30-40 mL 5% formic acid to the digestion mixture, and incubating at 377C for 30 min, followed by direct desalting. Desalting was performed with m-tips [14] containing Oligo R3-material (PerSeptive Biosystems, Framingham, MA, USA). The detailed protocols for in-gel digestion and desalting can also be found from our web-pages, http://www3.btk.utu.…”

Section: Protein Identificationmentioning

confidence: 99%

A proteome database of human primary T helper cells

et al. 2001

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We have established the first public database of human primary T helper cell proteome using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. For the database, CD4+ human T cells were activated with anti-CD3+anti-CD28 antibodies and metabolically labeled with [35S]methionine for 24 h. Cells were lysed and proteins were separated by 2-DE. About 1500 protein spots were detected in the resulting 2-DE gels with silver staining, and 2000 spots with autoradiography. We have identified 91 proteins from the 2-DE gels using peptide mass fingerprinting, and annotated them to our database. The identified proteins are also linked to SWISS-PROTand NCBI protein databases. Our database is available via the Internet at http://www3.btk.utu.fi:8080/Genomics/Proteomics/Database.

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Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry Sample Preparation Techniques Designed for Various Peptide and Protein Analytes

Cited by 144 publications

References 2 publications

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation

Graphite powder as an alternative or supplement to reversed-phase material for desalting and concentration of peptide mixtures prior to matrix-assisted laser desorption/ionization-mass spectrometry

A proteome database of human primary T helper cells

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