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Research: Xiaoning Lu and Haining Zhu Tube-Gel Digestion: A Novel Proteomic Approach for High Throughput Analysis of Membrane Proteins Mol Cell Proteomics 2005; 4: 1948-1958 [Abstract] [Full text] [PDF]

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The Response to Dr. Veenstra’s Letter to the Editor

Haining Zhu   (24 October 2005)

Letter regarding: Tube-Gel digestion: A novel proteomic approach for high throughput analysis of mem

Timothy D Veenstra, Thomas P. Conrads and Josip Blonder   (24 October 2005)

The Response to Dr. Veenstra’s Letter to the Editor 24 October 2005
Haining Zhu University of Kentucky Send letter to journal: Re: The Response to Dr. Veenstra’s Letter to the Editor haining{at}uky.edu Haining Zhu	We appreciate Dr. Veenstra’s letter. We cited Dr. Veenstra’s and another lab’s work for their credit that organic solvent can be used as an alternative to detergents to solubilize membrane proteins. We, by no means, intended to criticize their or anyone’s work or the solution-based membrane proteomics. We made a relatively general statement that the concerns of the cited articles “are the toxic solvents used as well as the compromised proteolytic enzymatic activity in organic solvents”. We appreciate that Dr. Veenstra agreed that methanol used in the study is toxic and that trypsin activity is reduced in 60% (v/v) methanol. To the credit of Dr. Veenstra again, their work avoided using a more toxic reagent CNBr that was used in the other reference cited together with theirs. We will clarify our previous statement to “The concerns of the above methods are the toxic reagents used (e.g. CNBr) as well as the compromised enzymatic activity in organic solvent”. Dr. Veenstra raised the issue of sequence coverage of bacteriorhodopsin in his letter. In our study, we injected 700 femto-mole (approximately 27 ng) of bacteriorhodopsin digestion peptides for LC-MS/MS analysis and yielded 21% sequence coverage using automatic MASCOT search without further manual hunting for other possible peptides. In Dr. Veenstra’s work, 5 l of sample with a final concentration of 0.4 g/l was subjected to the LC-MS/MS analysis. Based on the author’s statement that the H. halobium purple membrane contains a high abundance of bacteriorhodopsin, it is estimated that approximately 1 g bacteriorhodopsin was analyzed by LC-MS/MS in their study. It is not surprising that they obtained higher sequence coverage since they used approximately 30 to 40 times more proteins in the analysis. We acknowledge that the covalent modification of proteins by acrylamide is a real possibility for all polyacrylamide gel based protocols including ours. We have addressed this in the paper, see “It is noted that all 35 cysteine residues in BSA were identified in the carbamidomethyl form in the above experiments using the Tube-Gel protocol, suggesting that DTT reduction and IAA alkylation steps were highly effective in the Tube-Gel protocol.” It is also noted that 5 µl of 40% acrylamide was used to form a 20 l 10% poly-acrylamide gel matrix in our Tube-Gel digestion protocol, thus the potential toxicity of monomeric acrylamide is minimal. Haining Zhu Assistant Professor Department of Molecular and Cellular Biochemistry University of Kentucky
Letter regarding: Tube-Gel digestion: A novel proteomic approach for high throughput analysis of mem 24 October 2005
Timothy D Veenstra, Director Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., Frederick MD, Thomas P. Conrads and Josip Blonder Send letter to journal: Re: Letter regarding: Tube-Gel digestion: A novel proteomic approach for high throughput analysis of mem veenstra{at}ncifcrf.gov Timothy D Veenstra, et al.	In their manuscript entitled "Tube-Gel digestion: A novel proteomic approach for high throughput analysis of membrane proteins" Lu and Zhu make a direct comparison of their technique to solubilize membrane proteins with a method that we published in 2005 (ref 15 in manuscript). Their criticism of our method, as described in the introduction, refers to the use of "the toxic substances used and the compromised proteolytical enzymatic activity in organic solvents". While our study did show that trypsin activity is reduced in 60% (v/v) methanol, it also showed that the activity was sufficient to provide complete digestion of membrane proteins in this solvent. In fact, our study showed 100% coverage of bacteriorhodopsin (including modifications) while the study by Lu and Zhu only attained 21% coverage. Another criticism of our procedure is the use of the toxic solvent methanol. Compared to the toxicity of acrylamide, which is used in the study by Lu et al., the toxicity of methanol is minor not only based on dose per kg body weight but also the fact that methanol needs to be ingested to be toxic while acrylamide is easily inhaled. Furthermore, no mention was made in the paper by Lu et al. regarding the facile ability of acrylamide monomer to covalently modify proteins, hence, unnecessarily complicating downstream bioinformatic analyses. Unfortunately the authors of this paper have utilitized unfounded criticisms of our work in order to substantiate what they have accomplished. While their study has merit, the authors should refrain from trying to present it in a better light through unfounded criticisms of other scientist's work.