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>
	<li>Hao, P., Adav, S. S., Gallart-Palau, X., 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=Recent+advances+in+mass+spectrometric+analysis+of+protein+deamidation.">Recent advances in mass spectrometric analysis of protein deamidation.</a> Mass Spectrom Rev. , (2016).</li><li>Geiger, T., Clarke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deamidation,+isomerization,+and+racemization+at+asparaginyl+and+aspartyl+residues+in+peptides.+Succinimide-linked+reactions+that+contribute+to+protein+degradation.">Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation.</a> J Biol Chem. 262 (2), 785-794 (1987).</li><li>Serra, A., Gallart-Palau, X., Wei, J., Sze, S. 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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. 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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 height="21" style="height:21px;width:359px;">Bullet Blender</td>
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			<td style="width:424px;">Eppendorf&#160;</td>
			<td>T9661-1000EA</td>
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			<td style="width:424px;">Hettich Zentrifugen</td>
			<td style="width:207px;">Rotina 380 R</td>
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			<td height="21" style="height:21px;width:359px;">Phosphate buffer solution 10X (diluted to 1x)</td>
			<td style="width:424px;">Sigma-Aldrich</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>
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			<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 ...

The overall goal of this procedure is to use shotgun proteomics to characterize and quantify endogenous glutamine and asparagine deamidation products in complex proteomes. This method can help answer key questions in biochemistry, such as the influence of a spontaneous and antimatic processors on the isometric product gluteo of glutamine and asparagime deamination. The main advantage of this technique is that glutamine deamination isomers are separated on a long length ionic change capillary column, maximizing the ability to separate peptides by the isomatic points.To begin column construction, suspend 50 milligrams of weak anion exchange packing material in 3.5 milliliters of 90%isopropanol, in water. Secure female to female fitting, a microferal, and a female nut at one end of a 50 centimeter length of peak tubing with an inner diameter of 200 micrometers. Fit a 1/16th inch screen with one micrometer pores at the end of the tubing inside the microferal.Using pressure pump set to 4, 500 psi, pack the anion exchange into the capillary until the material is visible at the top. Attach another female-to-female fitting, microferal, and female nut to the top of the packed capillary at the opposite end to finish assembling the column. Wash 50 to 100 milligrams of tissue with phosphate buffered saline for five minutes.Repeat this wash twice, and then place the washed tissue in tubes with safety caps. Add to each tube one weight equivalent of stainless steel beads, and 2.5 equivalents by volume of an aqueous 100 millimolar solution of ammonium acetate with 1%sodium deoxycholate. Homogenize the tissue at maximum intensity for five minutes at four degrees Celsius.Then, centrifuge the homogenate for ten minutes at 10, 000 x g and four degrees Celsius. Transfer the supernatant to a 1.5 milliliter tube. Continue homogenizing and centrifuging the tissue pellet, combining the supernatants each time until no pellet is observed.Quantify the protein concentration in the combined supernatants with a bicinchoninic acid assay. Next, add to the homogenate a one molar solution of dithiothreitol in 100 millimolar ammonium acetate to attain a ten millimolar dithiothreitol concentration in the sample. Incubate the homogenate in a water bath at 60 degrees Celsius for thirty minutes.Add to the homogenate a one molar solution of iodoacetamide in 100 millimolar ammonium acetate to attain a 20 millimolar iodoacetamide concentration in the sample. Incubate the homogenate at room temperature in the absence of light for 45 minutes. Then dilute the homogenate with two volume equivalents of a ten millimolar solution of dithiothreitol in 100 millimolar ammonium acetate.Incubate the homogenate at 37 degrees Celsius for 30 minutes. Add to the homogenate a sufficient volume of sequencing grade modified trypsin in 100 millimolar ammonium acetate to achieve a 1 to 50 protein enzyme ratio by weight in the sample. Incubate the sample at 30 degrees Celsius overnight to digest the homogenate.Quench the digestion with 0.5%of the sample weight of formic acid, precipitating the sodium deoxycholate salts. Gently vortex the samples containing SDC salts and then centrifuge the samples for ten minutes at 12, 000 x g and four degrees Celsius. Decant the supernatant into a new tube, being careful not to disturb the pellet of SDC salts.Redissolve the salts in a 0.5%by volume aqueous solution of ammonium hydroxide, vortexing vigorously. Condition a one gram C-18 sorbent cartridge with 5 milliliters of acetonitrile, followed by five milliliters of 0.1%trifluoroacetic acid in water. Then, load the digested sample onto the cartridge.Wash the samples three to five times with five milliliter portions of 0.1%TFA. Then dilute the peptides with five milliliters of an aqueous solution of 75%acetonitrile, and 0.1%formic acid. Dry the fluid in a vacuum concentrator.Add 200 microliters of 75%acetonitrile, with 0.1%formic acid to the residue. Vortex the mixture for at least ten minutes, and then sonicate the mixture for at least 30 minutes to reconstitute the dry peptides. Dilute the sample as needed, so that the injected sample contains one to three micrograms of protein.Connect the LAX capillary column to an ultra high pressure liquid chromatography instrument. Prepare 0.1%formic acid solutions in water and acetonitrile. Set the flow rate to 0.4 microliters per minute, and set up a 1, 200 minute gradient run.Configure the mass spectrometer scan events to alternate data acquisition, between full Fourier transform mass spectrometry and Fourier transform tandem mass spectrometry. Perform LERLIC MS/MS to separate and characterize the peptides. Use the resulting data and proteomic software to perform a protein database search.Export the database search results to spreadsheet software. Identify and extract the list of deamidated peptides and the corresponding non-deamidated peptides. Extract the ion chromatagrams of each of these peptides with five parts per million of mass tolerance.Inspect the extracted ion chromatagrams for separated double peak illusion of isomeric products. Using LERLIC MS/MS, deamidated isoforms of glutamine and asparagine were separated and identified from a protein sample at two different retention times. Enzymatically modified intermediate glutamine residues of transamidation showing an inverted gamma alpha glutamil ratio were identified.The succinimide intermediate in asparagine residues was also identified with this method. As the mildly acidic sample processing conditions stabilize the succinimide intermediate. This suggests that the LERLIC MS/MS technique can be applied to proteome wide study of succinimide intermediates.After its development, this technique paved the way for researchers in biochemistry to characterize protein deamidation in contexts ranging from archeology to biomedical research.

lerlic-msms-for-depth-characterization-quantification-glutamine

Research

JoVE Journal

Biochemistry

8.0K Views.  Nanyang Technological University. The overall goal of this procedure is to use shotgun proteomics to characterize and quantify endogenous glutamine and asparagine deamidation products in complex proteomes. This method can help answer key questions in biochemistry, such as the influence of a spontaneous and antimatic processors on the isometric product gluteo of glutamine and asparagime deamination. The main advantage of this technique is that glutamine deamination isomers are separated on a long length ionic change capillary ...