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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Research

JoVE Journal

Biochemistry

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.
The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (&#8805;42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

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 ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...