Name: a spin-tip enrichment strategy for simultaneous analysis of n-glycopeptides and phosphopeptides from human pancreatic tissues
Uploaded: 2022-05-04T14:00:00
Description: Watch this Scientific Journal Video about A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues at JoVE.com

This research was supported in part by grant funding from the NIH (R01DK071801, RF1AG052324, P01CA250972, and R21AG065728), and Juvenile Diabetes Research Foundation (1-PNF-2016-250-S-B and SRA-2016-168-S-B). Data presented here were also in part obtained through support from an NIH/NCATS UL1TR002373 award through the University of Wisconsin Institute for Clinical and Translational Research. The Orbitrap instruments were purchased through the support of an NIH shared instrument grant (NIH-NCRR S10RR029531) and Office of the Vice Chancellor for Research and Graduate Education at the University of Wisconsin-Madison. We would also like to acknowledge the generous support of the University of Wisconsin Organ and Tissue Donation Organization who provided human pancreas for research and the help of Dan Tremmel, Dr. Sara D. Sackett, and Prof. Jon Odorico for providing the samples to our lab. Our research team would like to give special thanks to the families who donated tissues for this study. L.L. acknowledges NIH grant S10OD025084, a Pancreas Cancer Pilot grant from the University of Wisconsin Carbone Cancer Center (233-AAI9632), as well as a Vilas Distinguished Achievement Professorship and the Charles Melbourne Johnson Distinguished Chair Professorship with funding provided by the Wisconsin Alumni Research Foundation and University of Wisconsin-Madison School of Pharmacy.

Consent was obtained for the use of pancreatic tissues for research from the deceased&#39;s next of kin and an authorization by the University of Wisconsin-Madison Health Sciences Institutional Review Board was obtained. IRB oversight is not required because it does not involve human subjects as recognized by 45 CFR 46.102(f).
CAUTION: Care should be taken when handling the reagents used in this protocol, which include acids (formic, acetic, trifluoroacetic), bases (ammonium hydroxide), and cr...

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The dual-functional Ti(IV)-IMAC strategy is useful for the simultaneous analysis of N-glycopeptides and phosphopeptides from the same sample in a single sample preparation workflow. ERLIC-based methods have also been shown to perform simultaneous enrichment of PTMs. Both strategies have been used previously for deep coverage in PTM analyses14,18. In adapting the dual Ti method to decreasing sample incubation time by using spin-tips, we hope that this protocol has...

Protein post-translational modifications (PTMs) play a major role in modulating protein structures and consequently their functions and downstream biological processes. The diversity of the human proteome increases exponentially due to the combinatorial variability afforded by various PTMs. Different variants of proteins from their canonical sequences as predicted by the genome are known as proteoforms, and many proteoforms arise from PTMs1. Studying proteoform diversity in health and disease has become an area of research of great interest in recent years2,3.
The study of proteoforms and more specifically PTMs with great depth has become more facile through the development of mass spectrometry (MS)-based proteomics methods. Using MS, analytes are ionized, fragmented, and identified based on the m/z of fragments. Enrichment methods are often necessary due to the low relative abundance of PTMs compared to non-modified forms of proteins. Though analysis of intact proteins and their PTMs, called top-down analyses, have become more routine, the enzymatic digestion of proteins and the analysis of their component peptides in bottom-up analyses is still the most widely used route for PTM analysis. The two most widely studied PTMs, and the two most common PTMs in vivo, are glycosylation and phosphorylation4. These two PTMs play major roles in cell signaling and recognition and thus are important modifications to characterize in disease research.
The chemical properties of various PTMs often provides routes toward enrichment of these PTMs at the protein and peptide levels prior to analysis. Glycosylation is a hydrophilic PTM due to the abundance of hydroxyl groups on each monosaccharide. This property can be used to enrich glycopeptides in hydrophilic interaction chromatography (HILIC), which can separate more hydrophilic glycopeptides from the hydrophobic non-modified peptides5. Phosphorylation adds the phosphate moiety, which is negatively charged except at acidic pH. Due to this charge, various metal cations, including titanium, can be used to attract and bind phosphopeptides while non-phosphorylated species are washed away. This is the principle of immobilized metal affinity chromatography (IMAC). Further discussions of these and other enrichment strategies for glycosylation and phosphorylation can be found in recent reviews6,7.
Comparatively large amounts of starting peptide material (0.5 mg or more) are often needed for enrichment protocols due to the low stoichiometry of PTMs on peptides. In scenarios where this amount of sample may not be easily obtained, such as tumor core biopsy or cerebrospinal fluid analyses, it is beneficial to use facile workflows that result in maximum biomolecular information. Recent strategies developed by our lab and others have highlighted the simultaneous and parallel analysis of glycosylation and phosphorylation using the same PTM enrichment workflow8,9,10,11,12. Though the chemical properties of these two PTMs may differ, these PTMs may be analyzed in multiple steps due to the innovative separation techniques and materials used. For example, electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) overlays separations based on hydrophilic interactions between analytes and the mobile phase with charge-charge interactions between analytes and the stationary phase material13,14,15,16. At acidic pH, the attraction of phosphorylated peptides to the stationary phase can improve their retention and separation from non-modified peptides. Material consisting of Ti(IV) immobilized on hydrophilic microspheres can be used for HILIC and IMAC-based elution to separate phosphopeptides and neutral, acidic, and mannose-6-phosphorylated glycopeptides17,18. This strategy is known as dual-functional Ti(IV)-IMAC. Using these strategies for enriching multiple PTMs in a single workflow can make analyses of potential PTM crosstalk interactions more accessible. Additionally, the total sample amount and time requirements are less than the conventional enrichment methods when performed in parallel (i.e., HILIC and IMAC on separate sample aliquots).
To demonstrate the dual-functional Ti(IV)-IMAC strategy for simultaneous analysis of protein glycosylation and phosphorylation, we have applied it to analyze post-mortem human pancreatic tissues. The pancreas produces both digestive enzymes and regulatory hormones, including insulin and glucagon. The pancreatic function is impaired in pancreatic disease. In diabetes, the regulation of blood sugar is affected, leading to higher levels of glucose in the blood. In pancreatitis, inflammation results from auto-digestion of the organ3. Changes in PTM profiles, including glycosylation and phosphorylation, may result, as is often the case, in other diseases.
Here, we describe a protocol for a spin-tip based simultaneous enrichment method, based on a dual-functional Ti(IV)-IMAC strategy, for N-glycopeptides and phosphopeptides derived from proteins extracted from pancreatic tissue. The protocol includes protein extraction and digestion, enrichment, MS data collection, and data processing, as can be seen in Figure 1. Representative data from this study are available via ProteomeXchange Consortium with identifier PXD033065.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63735/63735fig01.jpg" /> 
Figure 1: Workflow for simultaneous analysis of N-glycopeptides and phosphopeptides from human pancreatic tissues. Tissues are first cryo-pulverized into a fine powder before protein extraction using the detergent sodium dodecyl sulfate (SDS). Proteins are then subjected to enzymatic digestion. The resulting peptides are aliquoted prior to enrichment using dual-functional Ti(IV)-IMAC. Raw data is collected using nanoscale reversed phase liquid chromatography-mass spectrometry (nRPLC-MS) and is analyzed using database searching software. <a href="https://www.jove.com/files/ftp_upload/63735/63735fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This protocol is intended to make PTM analyses more accessible and to enable more widespread analysis of multiple PTMs in the same workflow. This protocol can be applied to other complex biological matrices, including cells and biofluids.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic Acid, Glacial (Certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>A38S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone (Certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>A18-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, Optima LC/MS Grade</td>
			<td>Fisher Scientific</td>
			<td>A955-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate (Crystalline/Certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>A637-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide (Certified ACS Plus)</td>
			<td>Fisher Scientific</td>
			<td>A669-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Byonic software</td>
			<td>Protein Metrics</td>
			<td>n/a</td>
			<td>Commercial software used for glycoproteomic analysis (https://proteinmetrics.com/byos/)</td>
		</tr>
		<tr>
			<td>C18 BEH material</td>
			<td>Waters</td>
			<td>186002353</td>
			<td>Material removed from column and used to pack nano capillaries (pulledto integrate tip used directly in line with instrument inlet)</td>
		</tr>
		<tr>
			<td>CAE-Ti-IMAC, 100%</td>
			<td>J&#38;K Scientific</td>
			<td>2749380-1G</td>
			<td>Material used for dual-functional Ti(IV)-IMAC; can also be used for conventional IMAC/conventional phosphopeptide enrichment</td>
		</tr>
		<tr>
			<td>Cellcrusher kit</td>
			<td>Cellcrusher</td>
			<td>n/a</td>
			<td>Used for grinding tissue samples into powder before extraction</td>
		</tr>
		<tr>
			<td>Eppendorf 5424R Microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td>05-401-205</td>
			<td>For temperature-controlled centrifugation</td>
		</tr>
		<tr>
			<td>cOmplete protease inhibitor cocktail tablets</td>
			<td>Sigma</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT, Molecular Grade (DL-Dithiothreitol)</td>
			<td>Promega</td>
			<td>V3151</td>
			<td>Protein reducing agent</td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof (100%), USP</td>
			<td>Fisher</td>
			<td>22-032-601</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Analog Vortex Mixer</td>
			<td>Fisher Scientific</td>
			<td>02-215-414</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Low-Retention Microcentrifuge Tubes (1.5 mL)</td>
			<td>Fisher Scientific</td>
			<td>02-681-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Low-Retention Microcentrifuge Tubes (2 mL)</td>
			<td>Fisher Scientific</td>
			<td>02-681-321</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Model 120 Sonic Dismembrator</td>
			<td>Fisher Scientific</td>
			<td>FB120110</td>
			<td>For sample lysis using ultrasonication</td>
		</tr>
		<tr>
			<td>Formic Acid, 99.0+%, Optima LC/MS Grade</td>
			<td>Fisher Scientific</td>
			<td>A117-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Fused silica capillary (75 &#956;m inner diameter, 360 &#956;m outer diameter)</td>
			<td>Polymicro Technologies LLC</td>
			<td>100 m TSP075375</td>
			<td>For in-house pulled and packed columns with integrated emitter</td>
		</tr>
		<tr>
			<td>Hydrofluoric acid (48 wt. % in H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>339261-100ML</td>
			<td>Used for opening emitter of pulled capillary column</td>
		</tr>
		<tr>
			<td>Iodoacetamide, BioUltra</td>
			<td>Sigma</td>
			<td>I1149-5G</td>
			<td>Protein reducing reagent</td>
		</tr>
		<tr>
			<td>MaxQuant software</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Free software used for phosphoproteomic analysis (https://www.maxquant.org/)</td>
		</tr>
		<tr>
			<td>Multi-therm Shaker with heating and cooling</td>
			<td>Benchmark Scientific</td>
			<td>H5000-HC</td>
			<td>Heating block</td>
		</tr>
		<tr>
			<td>Oasis HLB 1 cc Vac Cartridge, 10 mg Sorbent per Cartridge, 30 &#181;m, 100/pk</td>
			<td>Waters</td>
			<td>186000383</td>
			<td>Larger-scale cartridge desalting for tryptic digests (loading capacity approximately up to 1 mg each)</td>
		</tr>
		<tr>
			<td>OMIX C18 pipette tips, 100 &#181;L tip, 10 - 100 &#956;L elution volume, 1 x 96 tips</td>
			<td>Agilent</td>
			<td>A57003100</td>
			<td>Smaller-scale packed pipette tip for desalting for enrichment elutions</td>
		</tr>
		<tr>
			<td>P-2000 Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-2000/F</td>
			<td>For pulling nano-capillary columns for LC-MS</td>
		</tr>
		<tr>
			<td>PhosSTOP phosphatase inhibitor tablets</td>
			<td>Sigma</td>
			<td>4906845001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Quantitative Colorimetric Peptide Assay</td>
			<td>Thermo Fisher Scientific</td>
			<td>23275</td>
			<td></td>
		</tr>
		<tr>
			<td>PolySAX LP (12 &#956;m, pore size 300 &#197;)</td>
			<td>PolyLC</td>
			<td>BMSX1203</td>
			<td>Material for strong anion-exchange chromatography used for ERLIC/conventional glycopeptide enrichment</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic (Crystalline/Certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure injection cell with integrated magnetic stirplate</td>
			<td>Next Advance</td>
			<td>PC77-MAG</td>
			<td>For packing nano-capillary columns with stationary phase up to 2500 psi limit</td>
		</tr>
		<tr>
			<td>Proteome Discoverer software</td>
			<td>Thermo Fisher Scientific</td>
			<td>n/a</td>
			<td>Commercial software for proteomics anaysis (with integrated database searching software nodes) and data visualization (https://www.thermofisher.com/us/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/multi-omics-data-analysis/proteome-discoverer-software.html)</td>
		</tr>
		<tr>
			<td>SpeedVac SC110 Vacuum Concentrator Model SC110-120</td>
			<td>Savant</td>
			<td>n/a</td>
			<td>Centrifugal vacuum concentrator for drying samples (under heat)</td>
		</tr>
		<tr>
			<td>SDS Solution, 10% Sodium Dodecyl Sulfate Solution, Molecular Biology/Electrophoresis</td>
			<td>Fisher Scientific</td>
			<td>BP2436200</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing Grade Modified Trypsin</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (Crystalline/Certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>TopTip, Empty, 10-200 &#181;L, Pack of 96</td>
			<td>Glygen Corporation</td>
			<td>TT2EMT.96</td>
			<td>Empty pipette tip with micron-sized hole used that can be used to pack chromatographic materials for enrichments, bundled with tube adapters</td>
		</tr>
		<tr>
			<td>Triethylammonium bicarbonate buffer (TEAB, 1 M, pH 8.5 (volatile))</td>
			<td>Sigma</td>
			<td>90360-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid, Reagent Grade, 99%</td>
			<td>Fisher Scientific</td>
			<td>60-017-61</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base (White Crystals or Crystalline Powder/Molecular Biology)</td>
			<td>Fisher Scientific</td>
			<td>BP152-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C Mix, Mass Spec Grade</td>
			<td>Promega</td>
			<td>V5071</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea (Certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>U15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, Optima LC/MS Grade</td>
			<td>Fisher Scientific</td>
			<td>W64</td>
			<td></td>
		</tr>
	</tbody>
</table>

a spin-tip enrichment strategy for simultaneous analysis of n-glycopeptides and phosphopeptides from human pancreatic tissues

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological matrices is often necessary due to their low stoichiometry in comparison to non-modified analytes. Most enrichment workflows of PTMs on peptides in bottom-up proteomics workflows, where proteins are enzymatically digested before the resulting peptides are analyzed, only enrich one type of modification. It is the entire complement of PTMs, however, that leads to biological functions, and enrichment of a single type of PTM may miss such crosstalk of PTMs. PTM crosstalk has been observed between protein glycosylation and phosphorylation, the two most common PTMs in human proteins and also the two most studied PTMs using mass spectrometry workflows. Using the simultaneous enrichment strategy described herein, both PTMs are enriched from post-mortem human pancreatic tissue, a complex biological matrix. Dual-functional Ti(IV)-immobilized metal affinity chromatography is used to separate various forms of glycosylation and phosphorylation simultaneously in multiple fractions in a convenient spin tip-based method, allowing downstream analyses of potential PTM crosstalk interactions. This enrichment workflow for glyco- and phosphopeptides can be applied to various sample types to achieve deep profiling of multiple PTMs and identify potential target molecules for future studies.

Representative mass spectrometry data, including raw files and search results, have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD03306522.
In this work, duplicate injection replicates were analyzed for each enrichment elution. Identifications made from both technical replicates were collated in the final analysis. Due to the semi-stochastic nature of data-dependent acquisition in picking peptid...

Watch this Scientific Journal Video about A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues at JoVE.com

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues.

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological ...

The dual-functional Titanium immobilized metal affinity chromatography strategy can enrich both N-glycopeptides and phosphopeptides in the same workflow, increasing biomolecular information from the same analysis. The main advantage of this enrichment is two separation mechanisms, which enable the simultaneous separation of N-glycopeptides and phosphopeptides in separate fractions for downstream mass spectrometry analysis. Using this enrichment method can improve detection of glycosylation and phosphorylation in complex biological samples like pancreatic tissues toward disease biomarker discovery such as in diabetes and cancer.Because this method relies on spin tips and centrifugation, it is important to control the centrifugation speed, and make sure that samples are free of particulates to prevent any clogging. To begin, pre-chill the parts of the tissue pulverizer that will come in contact with the pancreatic tissues, namely the chamber, pulverizer, and recovery spoon, along with any spatulas, in a polystyrene container. Next, transfer tissue pieces into the pre-chilled sample holder, and add a liquid nitrogen to the tissue until they are frozen.After placing the pulverizer into the chamber, strike it using a mallet five to ten times to crush the sample, then remove the pulverizer from the chamber and scrape off adherent tissue powder and pieces. Next, add a spoonful of liquid nitrogen to the chamber if tissues begin to melt, and repeat the pulverization process until a fine powder is obtained and portion the samples into approximately 100-milligram aliquots into pre-chilled tubes. After preparing the lysis buffer, dissolve one tablet each of protease and phosphatase inhibitor in 500 microliters of water, for a stock of 20 times the desired final concentration, and add the required volume of stock of each inhibitor to the lysis buffer to achieve the final inhibitor concentrations.After adding 600 microliters of lysis buffer to the tube, incubate at 95 degrees Celsius in the heating block for 10 minutes with shaking at 800 RPM, then remove the samples from the heating block and let them cool to room temperature. Next, sonicate the samples at 60-Watt energy for 45 seconds, using 15 second pulses with a 30-second rest in-between, and pellet the samples at 3000 times G for 15 minutes at four degrees Celsius. Add the supernatant to five times its volume of precipitation solvent.For example, 300 microliters of lysis buffer to 1.5 milliliters of precipitation solvent containing 50%acetone, 49.9%ethanol, and 0.1%acetic acid, then chill overnight at 80 degrees Celsius. Pellet the samples again at 3000 times G for 15 minutes at four degrees Celsius, and remove the supernatant, then wash the pellet by breaking up with a spatula and mix with the same amount of precipitation solvent. After centrifuging, air dry the sample pellet in a fume hood for 15 minutes, and store at 80 degrees Celsius until ready to proceed.First, re-suspend the protein pellet in 300 microliters of freshly-made digestion buffer containing 50 millimolar triethylammonium bicarbonate buffer and eight molar urea. To the protein in solution, add dithiothreitol to a final concentration of five millimolar, mix, and reduce at room temperature for one hour, then add iodoacetamide to a final concentration at 15 millimolar. Mix, and alkylate at room temperature for 30 minutes in the dark.Next, add Lys-C/Trypsin at 1 to 100 enzyme-to-protein ratio, and incubate at 37 degrees Celsius in an incubator for four hours, then add 50 millimolar triethylammonium bicarbonate buffer to dilute the eight molar urea used to less than one molar. After adding trypsin at 1 to 100 enzyme-to-protein ratio, incubate at 37 degrees Celsius in an incubator overnight, subsequently quench the digestion with the addition of trifluoroacetic acid. For every one milligram of starting protein, condition a desalting cartridge with one milliliter of acetonitrile, and thrice with one milliliter of 0.1%trifluoroacetic acid, then load the digested mixture onto the desalting cartridge, and wash the mixture thrice using one milliliter of 0.1%trifluoroacetic acid.Next, elute peptides using one milliliter of 60%acetonitrile and 0.1%formic acid solution, then dry eluted peptides at approximately 35 degrees Celsius using a centrifugal vacuum concentrator until the solvent has completely evaporated. After re-suspending the peptides in water, estimate peptide concentrations using a peptide assay according to the manufacturer's protocol, then portion the peptides into 500-microgram aliquots, and dry completely. Weigh approximately three milligrams of cotton wool and pack it into an empty spin tip.After transferring one gram of Titanium immobilized metal affinity chromatography material into a tube, add 0.1%trifluoroacetic acid to a known concentration of material. After adding enough slurry to a spin tip to transfer 20 milligrams of material, wash the spin tip by centrifuging with 200 microliters of 0.1%trifluoroacetic acid. Re-suspend the samples in 200 microliters of loading/washing solvent, and flow through the spin tip.After washing the spin tip by centrifugation with loading/washing solvent, wash the spin tip with 200 microliters of 80%acetonitrile 0.1 formic acid solution, then elute the peptides with 200 microliters of E1 and E2, and analyze each elution separately. Repeat the elution process using E3 and E4 solvents, and combine the appropriate elution fractions before downstream analysis. Before performing the elutions at basic pH, condition the material thrice using 200 microliters of 90%acetonitrile in 2.5%ammonium hydroxide, then elute the peptides with 200 microliters of E5 and E6 solvents, and analyze these elutions separately after desalting using a packed tip.Next, elute the peptides with 200 microliters of different concentrations of acetonitrile and ammonium hydroxide. Afterward, combine these elutions, and analyze as one sample, E7, after desalting using a packed tip. Annotated high-confidence MS/MS spectra from N-glycosylated and phosphorylated peptides were obtained with rich fragmentation of precursors, increasing confidence in the identification, as assigned by the database-searching software.Peptide identifications were found from samples enriched using the dual-functional Titanium immobilized metal affinity chromatography spin tip method over each elution fraction. The majority of glycopeptides elute in the first four fractions, while the majority of phosphopeptides elute in the last three fractions, lessening potential interferences in ionization. The glycoproteomics results from the dual titanium method were compared to an Ehrlich-glycopeptide-only enrichment, demonstrating that proportions of each type of glycan after binning into six categories based on their compositions, are similar between both methods.Phosphoproteomics results from the dual titanium method were compared to a conventional immobilized metal affinity chromatography phosphopeptide-only enrichment or using both methods. The majority of phosphorylated amino acids were identified as serine and threonine, with around 1%phosphorylated tyrosine. Properly constructing the spin tip is important to ensure smooth elution.Plug the cotton firmly into the tip, so that the enrichment material can be packed on top of the cotton. We have used the dual-functional titanium IMAC method for simultaneous characterization of glycosylation and phosphorylation in most tissues and the SARS-CoV-2 spike protein.

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