This research was generously funded by the NIH NCI IMAT Program Grant R33 (CA147988-02), the NIH NIGMS Graduate Training in Molecular Biotechnology at NC State Grant (T32GM008776), the US Dept. of Education GAANN Fellowship Program in Molecular Biotechnology at NC State Grant (P200A140020), the W.M. Keck Foundation, and North Carolina State University. Hen plasma was obtained with the assistance of Dr. James N. Petitte and Rebecca Wysocky in the NC State University Dept. of Poultry Science.

<ol>
	<li>Wisniewski, J. R., Zougman, A., Nagaraj, N., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+sample+preparation+method+for+proteome+analysis.">Universal sample preparation method for proteome analysis.</a> Nat. Meth. 6, 359-362 (2009).</li><li>Preston, R. J. S., Rawley, O., Gleeson, E. M., O'Donnell, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+the+role+of+carbohydrate+determinants+in+regulating+hemostasis:+insights+and+opportunities.">Elucidating the role of carbohydrate determinants in regulating hemostasis: insights and opportunities.</a> Blood. 121, 3801-3810 (2013).</li><li>Hasnain, S. Z., Gallagher, A. L., Grencis, R. K., Thornton, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+calculation+of+all+possible+oligosaccharide+isomers+both+branched+and+linear+yields+1.05+x+10(12)+structures+for+a+reducing+hexasaccharide:+The+isomer+barrier+to+development+of+single-method+saccharide+sequencing+or+synthesis+systems.">A calculation of all possible oligosaccharide isomers both branched and linear yields 1.05 x 10(12) structures for a reducing hexasaccharide: The isomer barrier to development of single-method saccharide sequencing or synthesis systems.</a> Glycobiology. 4, 759-767 (1994).</li><li>Zaia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+and+glycomics.">Mass spectrometry and glycomics.</a> Omics. 14, 401-418 (2010).</li><li>Hirabayashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lectin-based+structural+glycomics:+Glycoproteomics+and+glycan+profiling.">Lectin-based structural glycomics: Glycoproteomics and glycan profiling.</a> Glycoconjugate J. 21, 35-40 (2004).</li><li>Nilsson, J., 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=Enrichment+of+glycopeptides+for+glycan+structure+and+attachment+site+identification.">Enrichment of glycopeptides for glycan structure and attachment site identification.</a> Nat. 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H., Lilley, L. M., Enamorado, M. F., Comins, D. L., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+derivatization+of+N-linked+glycans+for+increased+ion+abundance+in+electrospray+ionization+mass+spectrometry.">Hydrophobic derivatization of N-linked glycans for increased ion abundance in electrospray ionization mass spectrometry.</a> J. Am. Soc. Mass. Spectr. 22, 1309-1317 (2011).</li><li>Baldwin, M. 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=Permethylation+and+tandem+mass+spectrometry+of+oligosaccharides+having+free+hexosamine:+Analysis+of+the+glycoinositol+phospholipid+anchor+glycan+from+the+scrapie+prion+protein.">Permethylation and tandem mass spectrometry of oligosaccharides having free hexosamine: Analysis of the glycoinositol phospholipid anchor glycan from the scrapie prion protein.</a> Anal. Biochem. 191, 174-182 (1990).</li><li>Harvey, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization+of+carbohydrates+for+analysis+by+chromatography;+electrophoresis+and+mass+spectrometry.">Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry.</a> J. Chromatogr. B. 879, 1196-1225 (2011).</li><li>Bradford, M. 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We have a collaborative relationship with Cambridge Isotope Laboratories (CIL) to disseminate the P2GPN reagent and technology to the broader scientific community in the form of a product (GTK-1000). However, we do not receive any revenue based on the sale of this product.

High-throughput quantitative methods are needed to facilitate routine glycan analysis. For the last thirty years, glycomics analysis has been limited to a subset of research groups, despite its importance in disease, clinical applications, and pharmaceuticals. The FANGS-P2GPN purification and tagging method for glycomics and proteomics performs the same analysis on a single aliquot of sample, reducing the cost of supplies and the amount of material needed (particularly important in human and mouse studies). Furthermore, efforts to minimize variability in preparations are critically important, as every additional step contributes to error, potentially masking important but low-abundant changes in case-control studies. Coupling of FANGS to hydrophobic hydrazide tagging allows protein and glycan samples to be run on the same RPLC column, enhances glycan ionization, provides for relative quantification, and can be quantitatively applied to plasma.
For N-glycan analysis, it is critical to use the suggested level of PNGase F to achieve full de-glycosylation. Though glycans are solvent exposed, denaturation of proteins and excess enzyme help ensure efficient and complete cleavage. For accurate quantitation of the glycans, it is necessary to ensure that they are completely dried after derivatization to quench the reaction and prevent cross-reactions when mixing the NAT and SIL species. Finally, when extending the workflow to glycosite analysis, timing of the steps is critical to minimize non-specific deamidation. The modified protocols provided for combined glycomics and proteomics analysis work consistently when performed accordingly.
The workflow achieves accurate relative quantitation of N-glycans from plasma compared to the gold-standard, SPE method. There is no apparent bias in the types of glycans extracted in terms of molecular weight, hydrophilicity, and compositional structure. Though we have not explored the qualitative analysis of O-linked glycans, we expect that FANGS could accommodate the addition of a &#946;-elimination step post-PNGase F digestion of N-linked glycans. However, procedures would require significant modification for reagent cleanup prior to mass spectrometry, and peptide analysis will be significantly impacted. For proteomics, the same depth of proteome coverage is achieved compared to traditional FASP methods. Importantly, methods achieve a minimal false discovery rate for N-glycan deamidation. While the method is compatible with 18O labeling of Asn during the PNGase digestion step22,23, the low glycosylation site false discovery rate suggests that it may not be necessary, further reducing costs and complexity.
The proteome is not enriched for glycoproteins in this method, which has both advantages and disadvantages. Certain low abundant glycoproteins may not be detected in the analysis. However, the occupancy of glycosylated sites per protein, can be compared between biological samples. Additionally, the error and bias introduced from lectin affinity purification or chemical enrichment is eliminated. In conclusion, coupling of FANGS to the individuality normalization when labeling with glycan hydrazide tags strategy results in a simplified, quantitative, high-throughput method for the tandem analysis of the glycome and proteome with great potential for application in clinical case-control studies.

In the field of proteomics, filter aided sample preparation (FASP) has been widely adopted for its ability to minimize the amount of starting material, decrease sample preparation artifacts, and maximize sample throughput1. However, such a method has yet to emerge and gain traction for the field of glycomics. Development of high-throughput, quantitative workflows are needed because of the integral role of glycosylation in biological defense and its modulation by cancer or diseases2,3. In mammals, N-glycans are composed of repeating saccharide units (hexoses (Hex), hexosamines (HexNac), sialic acids (NeuAc), and fucoses (Fuc)), which decorate a core structure (Hex3HexNac2)4 covalently bound to asparagine. Though the glycospace is considerably large when isomers are counted (&#62;1012), it is quite small on a composition basis and molecular weights typically range from 1,000-8,000 Da5. The compositional homogeneity of the class and the hydrophilicity of glycans pose unique challenges to purification, separation, and mass spectrometry (MS) workflows6.
Traditionally, N-glycans are digested from proteins or peptides by peptide-N-glycosidase F (PNGase F) and then enriched by lectin affinity chromatography7, captured by hydrazide beads8, or purified via solid phase extraction (SPE)9,10. While these methods are all highly effective, they introduce extra steps for desalting and limit the number of samples simultaneously processed. Over the last decade, a number of high-throughput platforms for glycomics have been proposed. Kim et al. published a semi-automated method using a vacuum-operated, SPE 96-well plate11. Alternatively, an affinity-filter method (N-glyco-FASP) was developed by the Mann group, which required the initial derivatization of the filter with a composite of lectins12. Lastly, the Thomas-Oates group proposed a semi-quantitative method, the Filter Aided N-Glycan Separation (FANGS), which exploited the narrow composition size of the glycospace13. Relying on molecular weight cut-off filters, small contaminants were first washed to waste and then the N-glycans were digested and eluted. Deglycosylated proteins remain on the filter in this protocol and can be subjected to in-line FASP.
Identification and quantification of glycans by electrospray ionization (ESI) MS requires off-line separations for (partial) resolution of isomers and derivatization for detection of low-abundant species. Labeling in the individuality when normalizing with glycan hydrazide tags strategy confers compatibility with reverse-phase liquid chromatography (RPLC)14,15. The 4-phenethyl-benzohydrazide (P2PGN) hydrophobic tag mediates the hydrophilicity of the glycans, enhancing ionization by, on average, four-fold16. Though other techniques, such as permethylation17 or amine-reactive tagging chemistries18, offer similar advantages, in the hydrazide reaction, glycans are reacted 1:1 stoichiometrically in facile conditions. Relative quantification is achieved by tandem analysis of samples derivatized with native (NAT) or 13C6 stabile isotope labels (SIL).
The following method evolves FANGS for plasma applications and couples it to the P2GPN hydrophobic tag for accurate relative quantification. Furthermore, it was designed to perform shot-gun proteomics, deamidation profiling, and quantitative glycomics on a single aliquot of sample, without compromising the integrity of the analyses.

<table><tbody><tr><td>Acetic Acid (50%)</td><td>Sigma Aldrich&amp;nbsp;</td><td>45754</td><td></td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH015-4</td><td></td></tr><tr><td>Ammonium Bicarbonate</td><td>Sigma Aldrich&amp;nbsp;</td><td>A6141</td><td></td></tr><tr><td>Bradford Reagent</td><td>Sigma Aldrich&amp;nbsp;</td><td>B6916</td><td>Alternative: Bicinchoninic acid kit (Sigma Aldrich BCA1)</td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich&amp;nbsp;</td><td>C1016</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 R</td><td>Alternate centrifuges that reach 14,000 x g are suitable</td></tr><tr><td>DL-Dithiothreitol, 1&amp;nbsp;M in solution</td><td>Sigma Aldrich&amp;nbsp;</td><td>646563</td><td></td></tr><tr><td>Easy-nLC 1000</td><td>Thermo Scientific</td><td>LC120</td><td>Alternate nano or ultra high pressure LCs will produce similar data, such as: 1. Dionex UltiMate&amp;Ograve; 3000 LC (Thermo Scientific); 2. Acquity UPLC (Waters).</td></tr><tr><td>Floating Tube Rack</td><td>TedPella</td><td>20831-20</td><td></td></tr><tr><td>Fetuin</td><td>New England Biolabs&amp;nbsp;</td><td>P6042S</td><td></td></tr><tr><td>Fisher Scientific Isotemp Standard Lab Ovens</td><td>Fisher Scientific</td><td>11-690-625F</td><td>Alternate incubators that reach 56&amp;nbsp;&amp;deg;C are suitable</td></tr><tr><td>Formic Acid</td><td>Sigma Aldrich&amp;nbsp;</td><td>56302</td><td></td></tr><tr><td>GE Microwave Oven</td><td>General Electric</td><td>57B5 E82904</td><td>Any microwave with adjustable power settings is suitable</td></tr><tr><td>INLIGHT Glycan Tagging Kit&amp;nbsp;</td><td>Cambridge Isotope Laboratories</td><td>GTK-1000</td><td>The INLIGHT kit provides NAT and SIL versions of the P2GPN reagent.</td></tr><tr><td>Iodoacetamide</td><td>Sigma Aldrich&amp;nbsp;</td><td>A3221</td><td></td></tr><tr><td>Kinetix 2.6 mM, 100 &amp;Aring;, C&lt;sub&gt;18&lt;/sub&gt; bulk stationary phase</td><td>Phenomenex&amp;nbsp;</td><td>Bulk Media</td><td>Alternative: Any C&lt;sub&gt;18&lt;/sub&gt; stationary phase 5&amp;nbsp;mM</td></tr><tr><td>Mascot Daemon Software and Server</td><td>Matrix Science</td><td></td><td>Alternative: Proteome Discoverer Software (Thermo Scientific)</td></tr><tr><td>Methanol, HPLC grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH230-4</td><td></td></tr><tr><td>PicoFrit Self-Pack Column: 360&amp;nbsp;&amp;#956;m, OD 75&amp;nbsp;&amp;#956;m ID, 15&amp;nbsp;&amp;#956;m tip, non-coated, 5 per box, 50 cm</td><td>New Objective</td><td>1 5 PF360-75-15-N-5</td><td></td></tr><tr><td>PNGase F (glycerol-free), 75,000&amp;nbsp;units/ml</td><td>New England BioLabs</td><td>P0705L</td><td></td></tr><tr><td>Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer</td><td>Thermo Scientific</td><td></td><td>Alternate high mass accuracy (5&amp;nbsp;ppm) mass spectrometers will provide similar data</td></tr><tr><td>RNase B</td><td>New England Biolabs&amp;nbsp;</td><td>P7817S</td><td></td></tr><tr><td>Trypsin from Porcine Pancreas</td><td>Sigma Aldrich&amp;nbsp;</td><td>T6567-5X</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich&amp;nbsp;</td><td>51456</td><td></td></tr><tr><td>Vacuum ConcentratorSavant SPD131DDA SpeedVac Concentrator</td><td>Thermo Scientific</td><td>SPD131DDA</td><td>Alternate vacuum concentrators are suitable</td></tr><tr><td>Vivacon 500 30 kDa Filters&amp;nbsp;</td><td>Sartorius Stedim Biotech</td><td>VN01H22</td><td>Alternative: Amicon Ultra 0.5 Centrifugal Filter Units with Ultracel-10&amp;nbsp;kDa Membrane (Millipore UFC501096)</td></tr><tr><td>Water, HPLC grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH365-4</td><td></td></tr><tr><td>Water, &lt;sup&gt;18&lt;/sup&gt;O</td><td>Cambridge Isotope Laboratories</td><td>OLM-240-97-1</td><td>The addition of &lt;sup&gt;18&lt;/sup&gt;O in the PNGase F digest step is optional and may not be necessary for deamidation studies completed with 95% confidence</td></tr><tr><td>Xcalibur 2.0</td><td>Thermo Scientific</td><td>XCALIBUR20</td><td></td></tr><tr><td>Zwittergent Test Kit</td><td>Merck Millipore</td><td>693030</td><td></td></tr></tbody></table>

a quantitative glycomics and proteomics combined purification strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

<img alt="Figure 1" src="/files/ftp_upload/53735/53735fig1.jpg" /> 
Figure 1: The scheme of the FANGS-P2GPN hydrophobic tagging coupled method (Method A) for combined proteomics and glycomics analysis is given. Steps that differ between glycomics-only processing and tandem glycomics and proteomics analysis, with glycosylation site identification, are highlighted. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The in-line proteomics and glycomics, filter-based P2GPN hydrophobic tagging method (Method A) was validated for identification, quantitation, and molecular weight bias using pooled hen plasma samples (Figure 1). For solely glycomics experiments, inter-comparisons in the abundances of N-glycans extracted by Method A to carbograph SPE (gold-standard, Method B) were made (Figure 2). There were no significant differences in the abundances of glycans between the two methods. The intra-variability was also assessed by quantifying the SIL: NAT ratio of glycans extracted by the same method. The log2-distributions of both methods were Gaussian, centered on zero, and not significantly different (Figure 3A-B). The molecular weight ranges between the two protocols completely overlapped, suggesting that the filter did not discriminate N-glycans based on molecular weight range and hydrophilicity (Figure 4).
<img alt="Figure 2" src="/files/ftp_upload/53735/53735fig2.jpg" /> 
Figure 2: An equimolar mixture of NAT and SIL N-glycans extracted by Method A or Method B was prepared from 2.5 &#956;l of hen plasma (N = 4). Samples were analyzed by UPLC-MS according to the recommended parameters suggested in section 6. The abundances for each glycan, in each NAT/SIL channel, were calculated by integrating the area under the extracted ion chromatogram (MMA = 3 ppm), correcting for the molecular weight overlap, and adjusting by the TGNF. These ratios are shown with their standard errors. The abundances of Method B: Method A N-glycans were not significantly different (p &#62;0.05). <a href="https://www.jove.com/files/ftp_upload/53735/53735fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53735/53735fig3.jpg" /> 
Figure 3: The intra-variability in the (A) Method A (N = 133) or (B) Method B (N = 123) strategies was compared. NAT and SIL equimolar mixtures of N-glycans, from the same extraction scheme, were analyzed over three technical replicates. The log2-distributions were centered on zero and were not significantly different between the two workflows. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53735/53735fig4.jpg" /> 
Figure 4: The molecular weight ranges of glycans from each strategy were compared. The two workflows yielded glycans spanning the same molecular weight range. N-glycans detected in one protocol versus the other fell just above the limit of detection (1 &#215; 105 abundance) and do not reflect systematic bias. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Retention of the deglycosylated proteins by the molecular weight filter enabled in-line proteome wide analysis and glycosylation site identification. Multiple protocols for the combined purification of glycans and proteins were compared to a traditional FASP preparation without deglycosylation (Table 5). Our typical 18 hr filter based PNGase F digestion, followed by FASP trypsin digest (Std protocol) resulted in significant levels of non-specific deamidation, which can interfere with the identification of glycosites. Therefore, a method utilizing a shorter incubation time at elevated temperature (50 &#176;C) and a PNGase F spike-in step (SPI protocol) was explored as an alternative, along with a microwave digestion protocol (MD protocol), to minimize non-specific deamidation. The glycans observed in the new digestion methods were not significantly different in compositions or abundances from the standard 18 hr, 37 &#176;C filter PNGase F digest (Figure 5). Combined with a short trypsin incubation, non-specific deamidation was significantly reduced, with a false positive rate for glycosites &#60;5% (Table 5).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2"></td>
			<td colspan="4">Proteins</td>
			<td></td>
			<td colspan="4">Peptides</td>
			<td></td>
			<td colspan="4">Deamidated Peptides</td>
			<td></td>
			<td colspan="4">Glycosites</td>
			<td></td>
			<td colspan="4">Glycoproteins</td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td colspan="2">+</td>
			<td colspan="2">-</td>
			<td></td>
			<td colspan="2">+</td>
			<td colspan="2">-</td>
			<td></td>
			<td colspan="2">+</td>
			<td colspan="2">-</td>
			<td></td>
			<td colspan="2">+</td>
			<td colspan="2">-</td>
			<td></td>
			<td colspan="2">+</td>
			<td colspan="2">-</td>
		</tr>
		<tr>
			<td colspan="2">FASP</td>
			<td colspan="2"></td>
			<td colspan="2">249</td>
			<td></td>
			<td colspan="2"></td>
			<td colspan="2">3083</td>
			<td></td>
			<td colspan="2"></td>
			<td colspan="2">795</td>
			<td></td>
			<td colspan="2"></td>
			<td colspan="2">5</td>
			<td></td>
			<td colspan="2"></td>
			<td colspan="2">5</td>
		</tr>
		<tr>
			<td colspan="2">18hr</td>
			<td colspan="2">217</td>
			<td colspan="2">304</td>
			<td></td>
			<td colspan="2">6013</td>
			<td colspan="2">5086</td>
			<td></td>
			<td colspan="2">1029</td>
			<td colspan="2">733</td>
			<td></td>
			<td colspan="2">257</td>
			<td colspan="2">24</td>
			<td></td>
			<td colspan="2">112</td>
			<td colspan="2">18</td>
		</tr>
		<tr>
			<td colspan="2">MD</td>
			<td colspan="2">270</td>
			<td colspan="2">266</td>
			<td></td>
			<td colspan="2">5190</td>
			<td colspan="2">5125</td>
			<td></td>
			<td colspan="2">465</td>
			<td colspan="2">455</td>
			<td></td>
			<td colspan="2">254</td>
			<td colspan="2">8</td>
			<td></td>
			<td colspan="2">102</td>
			<td colspan="2">7</td>
		</tr>
		<tr>
			<td colspan="2">SPI</td>
			<td colspan="2">281</td>
			<td colspan="2">288</td>
			<td></td>
			<td colspan="2">4729</td>
			<td colspan="2">4482</td>
			<td></td>
			<td colspan="2">602</td>
			<td colspan="2">573</td>
			<td></td>
			<td colspan="2">232</td>
			<td colspan="2">10</td>
			<td></td>
			<td colspan="2">145</td>
			<td colspan="2">8</td>
		</tr>
	</tbody>
</table>
Table 5: Comparisons of proteomic data from a standard trypsin digest versus the in-line FANGS-P2GPN tagging method were made. Preparations were performed with (+) and without (-) PNGase F to determine background rates of non-specific deamidation and estimate glycosite false positive rates. Proteins were identified with 1% false discovery rate (FDR); peptides were filtered based on &#34;high&#34; peptide confidence in Proteome Discoverer 1.4 (q &#60;0.01); glycosites were identified based on unique sequences containing deamidation of the conserved glycosylation motif (N-X-S/T); and glycoproteins were defined as identified proteins containing at least one identified glycosite.
<img alt="Figure 5" src="/files/ftp_upload/53735/53735fig5.jpg" /> 
Figure 5: Shortened protocols for glycans were developed to minimize deamination in the samples and compared to the 18hr FANGS PNGase F digest. The average differences in abundances were 10% and 5% for the MD (2.2.3) and SPI (2.2.2) protocols, respectively. Of the 48 glycans identified, 90% displayed less than 1.5-fold variation.&#160; <a href="https://www.jove.com/files/ftp_upload/53735/53735fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Quantitative Glycomics and Proteomics Combined Purification Strategy at JoVE.com

A Quantitative Glycomics and Proteomics Combined Purification Strategy

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

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Research

JoVE Journal

Chemistry

14.8K Views.  North Carolina State University. A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

Video: A Quantitative Glycomics and Proteomics Combined Purification Strategy

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Biochemistry

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Biology

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end approaches to isolate, enrich, and process proteins ahead of MS are critical for successful proteome characterization. Low, inconsistent protein recovery and residual impurities such as surfactants are detrimental to MS analysis. Protein precipitation is often considered unreliable, time-consuming, and technically challenging to perform compared to other sample preparation strategies. These concerns are overcome by employing optimal protein precipitation protocols. For acetone precipitation, the combination of specific salts, temperature control, solvent composition, and precipitation time is critical, while the efficiency of chloroform/methanol/water precipitation depends on proper pipetting and vial manipulation. Alternatively, these precipitation protocols are streamlined and semi-automated within a disposable spin cartridge. The expected outcomes of solvent-based protein precipitation in the conventional format and using a disposable, two-stage filtration and extraction cartridge are illustrated in this work. This includes the detailed characterization of proteomic mixtures by bottom-up LC-MS/MS analysis. The superior performance of SDS-based workflows is also demonstrated relative to non-contaminated protein.

The present protocol describes solvent-based protein precipitation under controlled conditions for robust and rapid recovery and purification of proteome samples prior to mass spectrometry.

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end ...

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Cancer Research

Automated Lectin-Based Glycan Profiling Using Bead Array in a Single Tip (BIST)

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of &lt;10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

Author Spotlight: Advancing Protein Glycosylation Research Using a Fully Automated System

A system for the automated and rapid analysis of glycans on proteins was previously developed. This article presents the protocol for "glyco-qualitative analysis," optimized for a broad spectrum of users, such as those engaged in analyzing glycan structures in biopharmaceuticals and other glycoconjugate materials.

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

A Quantitative Glycomics and Proteomics Combined Purification Strategy

Summary

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Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Glycopeptide Capture for Cell Surface Proteomics