Name: lerlic-ms/ms for in-depth characterization and quantification of glutamine and asparagine deamidation in shotgun proteomics
Uploaded: 2017-04-09T15:00:00
Description: Watch this Scientific Journal Video about LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics at JoVE.com

This work was in part supported by grants from the Singapore Ministry of Education (Tier 2: Grant ARC9/15), National Medical Research Council of Singapore (NMRC-OF-IRG-0003-2016), and NTU-NHG Ageing Research Grant (Grant ARG/14017). We would like to express our gratitude and most sincere thanks to Dr. Andrew Alpert and PolyLC team for kindly provided us with the packing materials that made possible this study.

1. Packing the Long-length Anion-exchange (LAX) Capillary Column
(Note: Although the LAX column can be in-home packed as we describe in this protocol, LAX columns are also commercially available, see Table of Materials and Reagents for further details).
<ol>
	<li>Suspend 50 mg of weak anion exchange packing material in 3.5 mL of packing buffer (Table 1) to prepare the slurry.</li>
	<li>Assemble the end of the capillary column (50 cm length - 200 &#1...

<ol>
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E., Robinson, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+clocks.">Molecular clocks.</a> Proc Natl Acad Sci U.S.A. 98 (3), 944-949 (2001).</li><li>Hao, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+separation+and+characterization+of+deamidated+peptides+with+RP-ERLIC-based+multidimensional+chromatography+coupled+with+tandem+mass+spectrometry.">Enhanced separation and characterization of deamidated peptides with RP-ERLIC-based multidimensional chromatography coupled with tandem mass spectrometry.</a> J Proteome Res. 11 (3), 1804-1811 (2012).</li><li>Li, X., Lin, C., O'Connor, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+deamidation:+differentiation+of+glutamic+acid+and+gamma-glutamic+acid+in+peptides+by+electron+capture+dissociation.">Glutamine deamidation: differentiation of glutamic acid and gamma-glutamic acid in peptides by electron capture dissociation.</a> Anal Chem. 82 (9), 3606-3615 (2010).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal Biochem. 150 (1), 76-85 (1985).</li><li>Serra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+proteome+coverage+is+increased+by+unique+peptide+recovery+from+sodium+deoxycholate+precipitate.">Plasma proteome coverage is increased by unique peptide recovery from sodium deoxycholate precipitate.</a> Anal Bioanal Chem. , 1-11 (2016).</li><li>Gallart-Palau, X., Serra, A., Sze, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncovering+Neurodegenerative+Protein+Modifications+via+Proteomic+Profiling.">Uncovering Neurodegenerative Protein Modifications via Proteomic Profiling.</a> Int Rev Neurobiol. 121, 87-116 (2015).</li><li>Liu, Y. D., van Enk, J. Z., Flynn, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+antibody+Fc+deamidation+in+vivo.">Human antibody Fc deamidation in vivo.</a> Biologicals. 37 (5), 313-322 (2009).</li><li>Serra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commercial+processed+soy-based+food+product+contains+glycated+and+glycoxidated+lunasin+proteoforms.">Commercial processed soy-based food product contains glycated and glycoxidated lunasin proteoforms.</a> Sci Rep. 6, 26106 (2016).</li><li>Serra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+peptidomic+strategy+to+decipher+the+molecular+diversity+of+cyclic+cysteine-rich+peptides.">A high-throughput peptidomic strategy to decipher the molecular diversity of cyclic cysteine-rich peptides.</a> Sci Rep. 6, 23005 (2016).</li><li>Leo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deamidation+at+asparagine+and+glutamine+as+a+major+modification+upon+deterioration/aging+of+proteinaceous+binders+in+mural+paintings.">Deamidation at asparagine and glutamine as a major modification upon deterioration/aging of proteinaceous binders in mural paintings.</a> Anal Chem. 83 (6), 2056-2064 (2011).</li><li>Wilson, J., van Doorn, N. L., Collins, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+extent+of+bone+degradation+using+glutamine+deamidation+in+collagen.">Assessing the extent of bone degradation using glutamine deamidation in collagen.</a> Anal Chem. 84 (21), 9041-9048 (2012).</li><li>Buszewski, B., Noga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+interaction+liquid+chromatography+(HILIC)--a+powerful+separation+technique.">Hydrophilic interaction liquid chromatography (HILIC)--a powerful separation technique.</a> Anal Bioanal Chem. 402 (1), 231-247 (2012).</li><li>Alpert, A. 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In this video-article we present a step-by-step protocol of LERLIC-MS/MS3, a method to perform in-depth characterization and to accurately determine the extent of protein deamidation and the enzymatic processes involved on this protein modification. LERLIC-MS/MS is based on the use of a long-length (50 cm) LAX under the principle of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)27. The use of a long-length column, as shown in our study<sup class="xre...

Deamidation is a protein posttranslational modification (PTM) that introduces a negative charge to the protein backbone through modification of asparagine (Asn) and/or glutamine (Gln) residues1. This modification while affecting Asn residues generates the isomeric products isoaspartic acid (isoAsp) and n-aspartic acid (Asp) at a common 3:1 ratio2. Notwithstanding, this ratio can be altered by the intervention of the repairing enzyme L-isoaspartyl methyltransferase (PIMT)3,4. Similarly, deamidation of Gln residues generates the isomeric gamma-glutamic acid (&#947;-Glu) and alpha-glutamic acid isoforms (&#945;-Glu) at an expected 1:7 ratio3,5, but this ratio can be shifted by the action of the ubiquitous enzyme transglutaminase 2 and other transglutaminases, including transglutaminase 1, an enzyme recently identified as associated with extracellular vesicles in the brain6.
The origin of deamidation can be either spontaneous or enzymatic, the former&#160;is especially common on Gln residues in which transglutaminases and other enzymes mediate&#160;inter/intra-molecular crosslinking via transamidation (see 3 for further details on Gln transamidation and its implications in several chronic and fatal human diseases). Therefore, deamidation is a PTM that has a crucial repercussion on the structure and function of affected molecules4,7,8 and requires an in-depth chemical characterization3 in the light of its diverse biochemical consequences including its service as molecular clock of aging9.
Although deamidation of Asn residues has been relatively well-characterized by bottom-up shotgun proteomics1,10, deamidation of Gln residues still does not have a suitable characterization method beyond the challenging analysis of model compounds by electron-based radical fragmentation11. We have recently developed a novel one-dimension shotgun proteomics strategy (LERLIC-MS/MS)3 that enables separation of Gln and Asn deamidation isoforms from complex biological samples and model compounds in a single analysis. LERLIC-MS/MS is based on the separation of tryptic digested peptides using a long-length (50 cm) ion exchange column (LAX) working on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) mode and coupled to tandem mass spectrometry (LC-MS/MS). This new analytical strategy has been used to characterize and relatively quantitate the extent of each deamidated residue in human brain tissues3. Nevertheless, the protocol outlined here will provide video imaging of LERLIC-MS/MS aimed to study the peculiarities of protein deamidation in the biochemical context of interest.
ETHICS STATEMENT
All procedures of this protocol have been approved by the institutional review board of the Nanyang Technological University in Singapore and have been performed in accordance to the institutional guidelines.

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			<td style="width:424px;">PolyLC Inc.</td>
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			<td height="21" style="height:21px;width:359px;">Sodium deoxycholate</td>
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			<td height="21" style="height:21px;width:359px;">Dithiothreitol</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Iodoacetamide</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">i6125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Formic acid</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">F0507 (HONEYWELL)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ammonium hydroxide</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">338818 (HONEYWELL)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Acetonitrile HPLC grade</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">675415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Isopropanol HPLC grade</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">675431</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Water HPLC grade</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">14263</td>
			<td></td>
		</tr>
	</tbody>
</table>

lerlic-ms/ms for in-depth characterization and quantification of glutamine and asparagine deamidation in shotgun proteomics

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in human pathology and other biochemical contexts. In order to perform characterization of protein deamidation, we have recently developed a novel long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method which can separate the glutamine (Gln) and asparagine (Asn) isoform products of deamidation from model compounds to highly complex biological samples. LERLIC-MS/MS is, therefore, the first shotgun proteomics strategy for the separation and quantification of Gln deamidation isoforms. We also demonstrate, as a novelty, that the sample processing protocol outlined here stabilizes the succinimide intermediate allowing its characterization by LERLIC-MS/MS. Application of LERLIC-MS/MS as shown in this video article can help to elucidate the currently unknown molecular arrays of protein deamidation. Additionally, LERLIC-MS/MS provides further understanding of the enzymatic reactions that encompass deamidation in distinct biological backgrounds.

Deamidation of Gln and Asn residues is considered a degenerative protein modification (DPM) implicated in several chronic and fatal diseases14. It has been demonstrated that this PTM can predict the half-life and degradative states of antibodies and other molecules in the human body and similar biological backgrounds1,15. The significance of protein deamidation, in fact, goes beyond the biomedical context, ...

Watch this Scientific Journal Video about LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics at JoVE.com

LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

Here we present a step-by-step protocol of the long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method. This is a novel methodology that enables for the first time quantification and characterization of the glutamine and asparagine deamidation isoforms by shotgun proteomics.

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in ...

Research

JoVE Journal

Biochemistry

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