Characterization of Hemoglobin Variants by MALDI-TOF MS Using a Polyurethane Membrane as the Sample Support

McComb, Mark E.; Oleschuk, Richard D.; Chow, A.; Ens, Werner; Standing, Kenneth G.; Perreault, Hélène; Smith, Moyra

doi:10.1021/ac9805753

Cited by 40 publications

(46 citation statements)

References 40 publications

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“…For high-throughput proteomics, the use of ZipTip C18 microcolumns becomes rather expensive in addition to being tedious. Regardless of the method of desalting, the peptide samples need to be concentrated after the removal of salt, and a number of eVective methods of concentrating peptide samples have been devised; these include the use of supports made of TeXon [5], polyethylene [6], polypropylene [7], and polyurethane [8,9]. An inexpensive MALDI sample support made with TeXon was recently described by Yuan and Desiderio [8] who showed that consistently good peptide mass spectra of trypsin-digested proteins were obtained using such plates.…”

mentioning

confidence: 99%

Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting

Tannu

Rao

et al. 2004

Analytical Biochemistry

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mentioning

confidence: 99%

Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting

Tannu

Rao

et al. 2004

Analytical Biochemistry

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“…On the other hand, MALDI-TOF MS gives single-charge peptide ions and has been used for identification of some singlemutation Hb variants (9,10 ). The present report shows that MALDI-TOF MS can be use to identify Hb variants.…”

Section: Clinical Chemistrymentioning

confidence: 99%

Identification by Mass Spectrometry of a Hemoglobin Variant with an Elongated β-Globin Chain

et al. 2005

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Among more than 800 hemoglobin (Hb) variants currently described in the HBVar database of the Globin Gene Server (1 ), variants with elongated chains are very rare. Standard protein techniques such as ion-exchange HPLC and isoelectric focusing (IEF) on polyacrylamide gel can detect many Hb variants (2 ), but correct identification of single mutated, inserted, or deleted amino acid residues requires more sophisticated techniques, such as electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (3-5 ). The interpretation of DNA sequencing in the presence of inserted nucleotide sequences in the heterozygous state can be difficult and requires direct and reverse sequencing. Protein analysis by MS can be used to check results from DNA sequencing and also can detect posttranslational changes such as acetylation (NH 2 terminus), deamidation, methionine oxidation, Hb addition products, or artifacts. ESI-MS and MALDI-TOF MS combined with specific tryptic digestion for peptide mass mapping are rapid and sensitive techniques to confirm inserted amino acid residues. We applied these techniques to the identification of a novel Hb variant with a five-amino acid insertion in the ␤-globin chain. These techniques allowed us to confirm the DNA sequencing results.Standard Hb analysis was performed by ion-exchange HPLC, IEF on polyacrylamide gel, and reversed-phase (RP)-HPLC of globin chains (6, 7 ). RP-HPLC was performed with a Vydac C 4 analytical column (The Separations Group) with CH 3 CN-H 2 O containing 1 mL/L trifluoroacetic acid (TFA) as the mobile phase (7 ). The same RP-HPLC system was also used for isolation of globin chains for MS studies. Electrospray experiments were performed on a SCIEX API 165 instrument (Applied Biosystems). Solutions were introduced by direct infusion at a flow rate of 5 L/min. Mass spectra were acquired at a 50-V orifice value in the positive ion mode, and the scan range was set at m/z 650-2150 Th (Thompson).For ESI-MS samples, we prepared a stock solution by diluting the whole blood 50-fold with water and desalting the stock solution by mixing with ϳ5 mg of AG 50W-X8 cation-exchange resin (Bio-Rad). We diluted 20 L of the desalted stock solution by adding 40 L of CH 3 OH-H 2 O (50:50 by volume) containing 1 mL/L HCOOH, and this solution was used for ESI-MS analysis. MALDI-TOF mass spectra were recorded on a Voyager DE-PRO mass spectrometer (Applied Biosystems) in the 700-5000 Da mass range. Use of a delayed extraction source and reflector instrumentation gave sufficient resolution to detect the monoisotopic peptide masses of [M ϩ H] ϩ ions. For MALDI-TOF sample preparation, we dried 0.5 mL of the 2-mL ␤-globin chain sample collected from RP-HPLC purification in a vacuum concentrator and redissolved the residue in 90 L of 50 mmol/L NH 4 HCO 3 buffer. This solution was digested with 10 L of trypsin solution (Promega; 0.1 g/L in 50 mmol/L NH 4 HCO 3 buffer) for 5 h at 37°C. We then dried 10 L of the digest so...

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“…McComb et al (McComb et al, 1997(McComb et al, , 1998Perreault et al, 2001) employed polyurethane (PU) as a platform to desalt protein samples. That research was based on a study by Oleschuk & Chow (1996), who showed that neutral species adsorb more strongly to PU than do charged species.…”

Section: E Polyurethanementioning

confidence: 99%

Non‐specific, on‐probe cleanup methods for MALDI‐MS samples

Bruening

Watson

2003

Mass Spectrometry Reviews

102

100

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High concentrations of contaminants such as salts and surfactants are often present in biological samples to solubilize or stabilize analytes such as proteins. Unfortunately, the presence of those contaminants often precludes direct analysis by MALDI-MS. Selective adsorption of analytes directly on modified MALDI probes, followed by rinsing to remove contaminants, overcomes this problem. This review focuses on various modifications of MALDI probes to allow the adsorption of proteins and DNA, even in a large excess of salt or surfactant. Interfaces deposited on the MALDI probes to adsorb analytes include films of commercial polymers, thin layers of matrix crystals, self-assembled monolayers, and ultrathin polymer films. Hydrophobic and ionic interactions both effect analyte adsorption on those interfaces, and patterned interfaces allow the concentration and purification of analyte molecules.

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Characterization of Hemoglobin Variants by MALDI-TOF MS Using a Polyurethane Membrane as the Sample Support

Cited by 40 publications

References 40 publications

Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting

Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting

Identification by Mass Spectrometry of a Hemoglobin Variant with an Elongated β-Globin Chain

Non‐specific, on‐probe cleanup methods for MALDI‐MS samples

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