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Accurate Mass Measurements Using MALDI-TOF with Delayed Extraction (1997)

Takach, Edward J. ; Hines, Wade M. ; Patterson, Dale H. ; [et al.]

Springer

The protein journal 16 (1997), S. 363-369

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ISSN: 1573-4943

Keywords: Accurate mass ; MALDI-TOF ; delayed extraction ; mass spectrometry ; peptide

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is now an essential tool in biopolymer analysis. Sensitivity and mass range are unsurpassed, but mass measurement accuracy and resolution have been limited. With delayed extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100–500). In database searching strategies, a smaller mass window, resulting from an increase in mass accuracy, greatly decreases the number of possible candidates. Mass measurement accuracy with errors less than 5 ppm is demonstrated on a mixture of 12 peptides ranging in mass from ca. 900 to 3700 Da. Mass measurements on 13 peaks in an unseparated tryptic digest of myoglobin gave results with an overall average error less than 3.5 ppm, with a maximum error of 7 ppm.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1026376403468

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Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: Implications for proteome mapping (1997)

Loo, Rachel R. Ogorzalek ; Mitchell, Charles ; Stevenson, Tracy I. ; [et al.]

Weinheim : Wiley-Blackwell

Electrophoresis 18 (1997), S. 382-390

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ISSN: 0173-0835

Keywords: Polyacrylamide gel ; Matrix-assisted laser desorption ; ionization ; Membranes ; Mass spectrometry ; Blotting ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of α- and β-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa α-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.

Additional Material: 6 Ill.