Virtual two‐dimensional gel electrophoresis of high‐density lipoproteins

Loo, Rachel R. Ogorzalek; Yam, Lang; Loo, Joseph A.; Schumaker, Verne N.

doi:10.1002/elps.200405955

Cited by 21 publications

(8 citation statements)

References 52 publications

(48 reference statements)

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“…In previous efforts [6, 7, 26, 29, 30], focused, washed IEF strips (Immobiline ™ DryStrip) had been incubated in saturated matrix dissolved in 50% ACN/49.9% H 2 O/0.1% TFA or 33.3% ACN/66.6% H 2 O/0.1% TFA ( i.e. , 1:1 or 1:2 matrix solution).…”

Section: Resultsmentioning

confidence: 99%

“…Available high repetition rate lasers and automation packages designed for MSI simplify and speed up data acquisition compared to our previous efforts in imaging IEF gels [6, 7, 26, 29, 30]. Now, a 4cm × 0.3cm section of a gel can be imaged in 5 min or less.…”

Section: Resultsmentioning

confidence: 99%

“…Gel strips were incubated for 15 minutes in a glass tray containing 200 mL of wash solution (49.9% H 2 O /50% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA)), mixing slowly on a lab rocker. The large volume of liquid removes or reduces the amounts of several substances essential to high-resolution electrophoresis but detrimental to MALDIMS:detergents, urea, thiourea, ampholytes, and residual mineral oil [26]. Despite this washing, CHAPS detergent clusters often appeared below m/z 5000 [26].…”

Section: Methodsmentioning

confidence: 99%

“…The large volume of liquid removes or reduces the amounts of several substances essential to high-resolution electrophoresis but detrimental to MALDIMS:detergents, urea, thiourea, ampholytes, and residual mineral oil [26]. Despite this washing, CHAPS detergent clusters often appeared below m/z 5000 [26]. After incubation, each gel was with drawn from the wash solution with tweezers and placed gel sideup atop a clean surface.…”

Section: Methodsmentioning

confidence: 99%

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Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics

Lohnes

Quebbemann

Liu

et al. 2016

Methods

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The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing (IEF) gel electrophoresis are imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by matrix-assisted laser desorption/ionization (MALDI) MS. Obtaining accurate intact masses from sub-picomole-level proteins embedded in 2D-PAGE gels or in IPG strips is desirable to elucidate how the protein of one spot identified as protein ‘A’ on a 2D gel differs from the protein of another spot identified as the same protein, whenever tryptic peptide maps fail to resolve the issue. This task, however, has been extremely challenging and is, in fact, rarely attempted. Virtual 2D gel/MS provides access to these intact masses. Modifications to our matrix deposition procedure improve the reliability with which IPG gels can be prepared; the new procedure is described. Development of this MALDI MS imaging (MSI) method for high-throughput with integrated ‘top-down’ MS to elucidate protein isoforms from complex biological samples is described and it is demonstrated that a 4-cm IPG gel segment can now be imaged in approximately 5 minutes. Gel-wide chemical and enzymatic methods with further interrogation by MALDIMS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MSI-based virtual 2D gel/MS platform may potentially link the benefits of ‘top-down’ and ‘bottom-up’ proteomics.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics

Lohnes

Quebbemann

Liu

et al. 2016

Methods

Self Cite

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show abstract

“…In addition, the perturbed electric field also increases the dispersion of the ions, which results in a loss of spectral resolution. These effects have been observed by different authors using high‐vacuum MALDI instruments with axial ionization sources 12–15. With single‐stage MALDI‐TOF instruments, this effect is not important and can be corrected by careful laser adjustment and internal or external calibration.…”

mentioning

confidence: 85%

Gold coating of non‐conductive membranes before matrix‐assisted laser desorption/ionization tandem mass spectrometric analysis prevents charging effect

Scherl

Zimmermann‐Ivol

Dio

et al. 2005

Rapid Comm Mass Spectrometry

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Acquisition of tandem mass spectra from peptides or other analytes deposited on non-conductive membranes is inhibited on instruments combining matrix-assisted laser desorption/ionization with tandem time-of-flight analyzers (MALDI-TOF/TOF) due to a charging effect. A thin layer of gold renders the membrane conductive. This allows adequate data acquisition on MALDI-TOF/TOF systems. Therefore, this methodology extends the capacity of the molecular scanner concept to tandem mass spectrometry.

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ApoM – A Novel Apolipoprotein with Antiatherogenic Properties

Nilsson‐Ehle

2007

High‐Density Lipoproteins

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Virtual two‐dimensional gel electrophoresis of high‐density lipoproteins

Cited by 21 publications

References 52 publications

Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics

Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics

Gold coating of non‐conductive membranes before matrix‐assisted laser desorption/ionization tandem mass spectrometric analysis prevents charging effect

ApoM – A Novel Apolipoprotein with Antiatherogenic Properties

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