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 cryogens (liquid nitrogen). Read the safety data sheets for the reagents used to become familiar with the associated hazards and needed precautions. Concentrations denoted using percentages are volume/total volume (v/v) and are diluted with water.
1. Tissue cryo-pulverization, lysis, and protein extraction
<ol>
	<li>Fill a dewar with liquid nitrogen. Pre-chill the parts of the tissue pulverizer that will come in contact with the pancreatic tissues, namely, the chamber, pulverizer, and recovery spoon in a polystyrene container.</li>
	<li>Transfer the frozen tissue pieces into the pre-chilled sample holder and add a spoonful of liquid nitrogen to the tissue.</li>
	<li>Place the pulverizer into the chamber and strike it using a mallet five to ten times to crush the sample. Remove the pulverizer from the chamber and scrape off adherent tissue powder and pieces.</li>
	<li>Add a spoonful of liquid nitrogen to the chamber if tissues begin to melt. Repeat the pulverization process until the sample is a fine powder without large tissue chunks. Portion the samples into approximately 100 mg aliquots into pre-chilled tubes.</li>
	<li>Prepare lysis buffer containing 4% sodium dodecyl sulfate (SDS), 150 mM NaCl, and 25 mM Tris (pH = 7.4). Dissolve one tablet each of protease and phosphatase inhibitor in 500 &#181;L of water for a 20x stock of each. Add the required volume of 20x stock of each inhibitor to the lysis buffer for a final 1x concentration.</li>
	<li>Add 600 &#181;L of lysis buffer per 100 mg tissue to the tube and incubate at 95 &#176;C in a heating block for 10 min with shaking at 800 rpm. Remove the samples from the heating block and let them cool to room temperature.</li>
	<li>Sonicate the samples at 60 W energy (20 kHz) for 45 s using 15 s pulses with a 30 s rest in between. Pellet the samples at 3,000 x g for 15 min at 4 &#176;C.</li>
	<li>Add the supernatant to a 5x precipitation solvent, 300 &#181;L of lysis buffer to 1.5 mL of precipitation solvent, containing 50% acetone, 49.9% ethanol, and 0.1% acetic acid. Chill overnight at -20 &#176;C.</li>
	<li>Pellet the samples again at 3,000 x g for 15 min at 4 &#176;C and remove the supernatant. Wash the pellet by breaking up with a spatula and mixing with the same amount of precipitation solvent. Pellet again, repeating the washing step 2x.</li>
	<li>Pellet the sample at 16,000 x g for 15 min at 4 &#176;C. Air dry the sample pellet in a fume hood for 15 min and store at -80 &#176;C until ready to proceed.</li>
</ol>
2. Protein digestion and desalting
<ol>
	<li>Resuspend the protein pellet in 300 &#181;L of freshly made digestion buffer containing 50 mM triethylammonium bicarbonate (TEAB) and 8 M urea.</li>
	<li>Estimate the protein concentration of the solution using a protein assay according to the manufacturer&#39;s protocol.</li>
	<li>To the protein in the solution, add dithiothreitol (DTT) to a final concentration of 5 mM, mix, and reduce at room temperature for 1 h. Then, add iodoacetamide (IAA) to a final concentration of 15 mM, mix, and alkylate at room temperature for 30 min in dark. Quench alkylation by repeating the addition of DTT in the same volume as before and mix.</li>
	<li>Add LysC/trypsin at 1:100 enzyme:protein ratio and incubate at 37 &#176;C for 4 h. Then, add 50 mM TEAB&#160;to dilute the 8 M urea used to &#60; 1 M. Add trypsin at 1:100 enzyme:protein ratio and incubate at 37 &#176;C overnight.</li>
	<li>Quench the digestion with the addition of trifluoroacetic acid (TFA) to 0.3% v/v. For every 1 mg of starting protein, condition a desalting cartridge (1 cc, 10 mg) with 1 mL of acetonitrile (ACN) and 3x with 1 mL of 0.1% TFA.</li>
	<li>Load the digested mixture onto the desalting cartridge. Wash the mixture 3x using 1 mL of 0.1% TFA. If the elution is slow, apply positive pressure but avoid a flow rate &#62; one drop per second.</li>
	<li>Elute peptides using 1 mL of 60% ACN and 0.1% formic acid (FA) solution. Dry eluted peptides at approximately 35 &#176;C&#160;using a centrifugal vacuum concentrator until the solvent has completely evaporated.</li>
	<li>Resuspend peptides in 300 &#181;L of water and estimate peptide concentrations using a peptide assay according to the manufacturer&#39;s protocol. Portion the peptides into 500 &#181;g aliquots and dry completely.</li>
</ol>
3. ERLIC N-glycopeptide enrichment
NOTE: Exact centrifuge speeds and times may differ based on samples and must be optimized. In general, 300 x g for 2 min is appropriate for conditioning and washing of the material and 100 x g for 5 min for eluting.
<ol>
	<li>Weigh approximately 3 mg of cotton wool and pack it into an empty 200 &#181;L pipette tip (spin-tip; see Table of Materials).</li>
	<li>Transfer strong anion-exchange enrichment material into a tube and add 200 &#181;L of 0.1% TFA per 10 mg material. For enrichment of 500 &#181;g of peptides, use 15 mg of the material. Activate the material by shaking for 15 min.</li>
	<li>Using a tube adapter, place the spin-tip over a 2 mL tube and add enough slurry to the tip for 15 mg of material. Remove the liquid by spinning in a benchtop centrifuge.</li>
	<li>Condition the material by centrifuging 3x with 200 &#181;L of ACN. Repeat the triplicate conditioning using 100 mM ammonium acetate (NH4Ac), 1% TFA, and 80% ACN/0.1% TFA (loading buffer).</li>
	<li>Resuspend 500 &#181;g peptide samples in approximately 200 &#181;L of loading buffer and flow through the spin-tip. Reload the flow-through 2x to ensure complete binding.</li>
	<li>Wash the material by centrifuging 5x using 200 &#181;L of 80% ACN/0.1% TFA, and then 2x using 200 &#181;L of 80% ACN/0.1% FA.</li>
	<li>Elute the peptides by centrifuging with 200 &#181;L of each of the following: 50% ACN/0.1% FA (E1); 0.1% FA (E2); 0.1% TFA (E3); 300 mM KH2PO4, 10% ACN (E4).</li>
	<li>Wash the material by centrifuging 2x with 200 &#181;L of 80% ACN/5% NH4OH. Elute the remaining peptides with 200 &#181;L of 10% NH4OH (E5).</li>
	<li>Dry all the elutions completely using a centrifugal vacuum concentrator at approximately 35 &#176;C.</li>
	<li>Perform desalting of the basic elution (E5) using a desalting tip according to manufacturer&#39;s protocol.</li>
	<li>Dry the elution from the desalting tip using a centrifugal vacuum concentrator at approximately 35 &#176;C to completeness.</li>
</ol>
4. Ti(IV)-IMAC phosphopeptide enrichment
NOTE: Exact centrifuge speeds and times may differ based on samples and must be optimized. In general, 300 x g for 2 min is appropriate for conditioning and washing of the material and 100 x g for 5 min for eluting.
<ol>
	<li>Weigh approximately 3 mg of cotton wool and pack it into an empty 200 &#181;L pipette tip (spin-tip).</li>
	<li>Transfer Ti-IMAC phosphopeptide enrichment material into a tube and add 200 &#181;L of 0.1% TFA per 10 mg material. For enrichment of 500 &#181;g peptides, use 10 mg of material.</li>
	<li>Using a tube adapter, place the spin-tip over a 2 mL tube and add enough slurry to the tip for 10 mg material. Remove the liquid by spinning in a benchtop centrifuge.</li>
	<li>Condition the material with 200 &#181;L of 40% ACN/3% TFA using the same centrifuge settings as described above. Resuspend peptide samples in 200 &#181;L of 40% ACN/3% TFA and flow through the spin-tip. Reload the flow-through twice to ensure more complete binding.</li>
	<li>Wash the material by centrifuging with 200 &#181;L of 50% ACN, 6% TFA, and 200 mM NaCl solution, followed by washing 2x in 200 &#181;L of 30% ACN and 0.1% TFA solution.</li>
	<li>Elute peptides with 200 &#181;L of 10% NH4OH. Dry the elution completely under vacuum. Perform desalting using a desalting tip according to the manufacturer&#39;s protocol. Dry the elution from the desalting tip under vacuum to completeness.</li>
</ol>
5. Dual-functional Ti(IV) simultaneous enrichment
NOTE: Exact centrifuge speeds and times may differ based on samples and must be optimized. In general, 300 x g for 2 min is appropriate for conditioning and washing of the material and 100 x g for 5 min for eluting.
<ol>
	<li>Weigh approximately 3 mg of cotton wool and pack it into an empty spin-tip.</li>
	<li>Transfer approximately 1 g of Ti(IV)-IMAC material into a tube. Add 0.1% TFA to a known concentration of material, e.g., 20 mg/200 &#181;L (material can be stored in this suspension at 4 &#176;C).</li>
	<li>For enrichment from 500 &#181;g peptides, add enough slurry to a spin-tip to transfer 20 mg of material. Wash the spin-tip by centrifuging with 200 &#181;L of 0.1% TFA.</li>
	<li>Resuspend the samples in 200 &#181;L of loading/washing solvent (80% ACN and 3% TFA) and flow through the spin-tip. Reload the flow-through 2x.</li>
	<li>Wash the spin-tip 6x by centrifuging with 200 &#181;L of loading/washing solvent. Wash the spin-tip with 200 &#181;L of 80% ACN/0.1% FA solution.</li>
	<li>Elute the peptides with 200 &#181;L of each of the following: 60% ACN/0.1% FA (E1) and 40% ACN/0.1% FA (E2). Analyze each elution separately.</li>
	<li>Elute the peptides with 200 &#181;L of each of the following: 20% ACN/0.1% FA and 0.1% FA. Combine the two elutions and analyze as one sample (E3).</li>
	<li>Elute the peptides with 200 &#181;L of each of the following: 40% ACN/3% TFA; 50% ACN/6% TFA, 200 mM NaCl; and 30% ACN/0.1% TFA. Combine these elutions and analyze as one (E4) after desalting using a packed tip.</li>
	<li>Condition the material to basic pH using 200 &#181;L of 90% ACN/2.5% NH4OH for 3x. Discard these washes.</li>
	<li>Elute the peptides with 200 &#181;L of each of the following: 60% ACN/10% NH4OH (E5) and 40% ACN/10% NH4OH (E6). Analyze these elutions separately after desalting using a packed tip.</li>
	<li>Elute the peptides with 200 &#181;L of each of the following: 20% ACN/10% NH4OH; 10% ACN/10% NH4OH; and 10% NH4OH. Combine these elutions and analyze as one (E7) after desalting using a packed tip.</li>
	<li>Dry all the elutions completely under vacuum.</li>
</ol>
6. Nano-flow reversed phase liquid chromatography-mass spectrometry (nRPLC-MS)
NOTE: MS data acquisition and analysis methods are diverse, and thus only one suggested LC-MS pipeline (and its associated parameters) is described here in the following steps. Samples generated using the previously outlined sample preparation and enrichment steps can be analyzed using other instrumental set-ups, including using commercially available chromatographic columns, given sufficient data quality.
<ol>
	<li>Pull and pack a 15 cm long capillary (75 &#181;m inner diameter) using C18 material as described in19. Prepare mobile phase A (0.1% FA in water) and mobile phase B (0.1% FA in ACN).</li>
	<li>Reconstitute enriched peptide samples in 15 &#181;L of 3% mobile phase B. Load 2 &#181;L of the sample (~13% sample volume) directly onto the column. Analyze each sample in technical duplicate.</li>
	<li>Elute peptides using a gradient from 3% to 30% mobile phase B over 90 min at a flow rate of 0.3 &#181;L/min. Wash the column using 75% B for 8 min followed by 95% B for 8 min, finishing the method with equilibration at 3% B for 12 min.</li>
	<li>Operate the mass spectrometer in the positive ion mode using data-dependent acquisition of the top 20 peaks with the following parameters: spray voltage 2 kV; MS1 detection in LC/MS from 400-2,000 m/z at 120,000 resolution, 2E5 automatic gain control (AGC) target, 100 ms maximum injection time, 30% RF lens, quadrupole isolation with 1.6 m/z window, for charge states 2-8 and undetermined, and 30 s dynamic exclusion; MS2 detection in the LC/MS using stepped HCD fragmentation at 22%, 30%, and 38%, fixed first mass 120 m/z at 30,000 resolution, 5E4 AGC target, and 2.5E4 minimum intensity requirement.</li>
</ol>
7. MS data analysis
NOTE: One data analysis pipeline using two different software to analyze the same dataset is presented here. Phosphorylation and glycosylation can be searched at the same time using a single software instead of two separate software as described here, though in general, software search time is proportional to the search space, i.e., number of PTMs considered. For this reason, two different software are used in parallel to the search for glycopeptides and phosphopeptides.
<ol>
	<li>Process raw data files using the appropriate software (see Table of Materials).
	<ol>
		<li>Search spectra against the UniProt human (or appropriate species-specific) database. Set carbamidomethylation of Cys as a fixed modification. Set the maximum number of missed enzymatic cleavages as two.</li>
	</ol>
	</li>
	<li>In the raw data files, using a commercial software (see Table of Materials), search for glycopeptides to be analyzed20. Use a 10 ppm precursor mass tolerance and a 0.01 Da fragment mass tolerance with a minimum peptide length of four residues.
	<ol>
		<li>Search using the software-embedded N-glycan database expanded with typical mannose-6-phosphate(M6P)glycans, including HexNAc(2)Hex(4-9)Phospho(1-2), HexNAc(3-4)Hex(4-9)Phospho(1-2), HexNAc(2)Hex(3-4)Phospho(1), and HexNAc(3)Hex(3-4)Phospho(1).</li>
		<li>Set N-glycosylation as a common modification. Set the maximum total modifications per peptide spectral match (PSM) as one common and two rare.</li>
		<li>Set the following variable modifications: oxidation of Met (rare), deamidation at Asn and Gln (rare), and phosphorylation at Ser, Thr, and Tyr (common). Set the following identification filters: score cut-off &#62; 150, |log prob| &#62; 1, and delta mod score &#62; 10.</li>
		<li>Export and analyze search results in the Proteins and Peptide Group tabs.</li>
		<li>Manually screen the identifications containing M6P glycans for the presence of the PhosphoHex oxonium ion (243 m/z) in the MS/MS spectra and remove false positive identifications, which do not contain this diagnostic ion.</li>
	</ol>
	</li>
	<li>In the raw data files, using an open-source software (see Table of Materials), search for phosphopeptides to be analyzed21. Use a 20 ppm first search peptide tolerance and a 4.5 ppm main search peptide tolerance.
	<ol>
		<li>Set the maximum number of modifications per peptide to 5. Set PSM and protein false discovery rate (FDR) to 1% and minimum peptide length to 7 residues.</li>
		<li>Set variable modifications as oxidation at Met, N-terminal protein acetylation, and phosphorylation at Ser, Thr, and Tyr. Analyze search results using the modificationSpecificPeptides files.</li>
	</ol>
	</li>
	<li>Determine the number of identifications of PTMs at the protein, peptide, and modification site levels.</li>
</ol>

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<li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metascape+provides+a+biologist-oriented+resource+for+the+analysis+of+systems-level+datasets%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Metascape provides a biologist-oriented resource for the analysis of systems-level datasets.</a> Nature Communications. 10 (1), 1523(2019).</li>
<li>Zacharias, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HILIC+and+ERLIC+enrichment+of+glycopeptides+derived+from+breast+and+brain+cancer+cells%5BTitle%5D)+AND+%22Journal+of+Proteome+Research%22%5BJournal%5D)">HILIC and ERLIC enrichment of glycopeptides derived from breast and brain cancer cells.</a> Journal of Proteome Research. 15 (10), 3624-3634 (2016).</li>
<li>Yang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+enrichment+methods+for+intact+N-+and+O-linked+glycopeptides+using+strong+anion+exchange+and+hydrophilic+interaction+liquid+chromatography%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Comparison of enrichment methods for intact N- and O-linked glycopeptides using strong anion exchange and hydrophilic interaction liquid chromatography.</a> Analytical Chemistry. 89 (21), 11193-11197 (2017).</li>
<li>Toghi Eshghi, S., Shah, P., Yang, W., Li, X., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GPQuest%3A+A+spectral+library+matching+algorithm+for+site-specific+assignment+of+tandem+mass+spectra+to+intact+N-glycopeptides%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">GPQuest: A spectral library matching algorithm for site-specific assignment of tandem mass spectra to intact N-glycopeptides.</a> Analytical Chemistry. 87 (10), 5181-5188 (2015).</li>
<li>Liu, M. -Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((pGlyco+2.0+enables+precision+N-glycoproteomics+with+comprehensive+quality+control+and+one-step+mass+spectrometry+for+intact+glycopeptide+identification%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification.</a> Nature Communications. 8 (1), 438(2017).</li>
<li>Lu, L., Riley, N. M., Shortreed, M. R., Bertozzi, C. R., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((O-Pair+Search+with+MetaMorpheus+for+O-glycopeptide+characterization%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">O-Pair Search with MetaMorpheus for O-glycopeptide characterization.</a> Nature Methods. 17 (11), 1133-1138 (2020).</li>
<li>Caval, T., Heck, A. J. R., Reiding, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Meta-heterogeneity%3A+Evaluating+and+describing+the+diversity+in+glycosylation+between+sites+on+the+same+glycoprotein%5BTitle%5D)+AND+%22Molecular+%26+Cellular+Proteomics%22%5BJournal%5D)">Meta-heterogeneity: Evaluating and describing the diversity in glycosylation between sites on the same glycoprotein.</a> Molecular & Cellular Proteomics. 20, 100010(2021).</li>
<li>Lee, J. S., Smith, E., Shilatifard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+language+of+histone+crosstalk%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The language of histone crosstalk.</a> Cell. 142 (5), 682-685 (2010).</li>
<li>Leutert, M., Entwisle, S. W., Villén, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decoding+post-translational+modification+crosstalk+with+proteomics%5BTitle%5D)+AND+%22Molecular+%26+Cellular+Proteomics%22%5BJournal%5D)">Decoding post-translational modification crosstalk with proteomics.</a> Molecular & Cellular Proteomics. 20, 100129(2021).</li>
<li>Hart, G. W., Slawson, C., Ramirez-Correa, G., Lagerlof, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cross+talk+between+O-GlcNAcylation+and+phosphorylation%3A+Roles+in+signaling%2C+transcription%2C+and+chronic+disease%5BTitle%5D)+AND+%22Annual+Review+of+Biochemistry%22%5BJournal%5D)">Cross talk between O-GlcNAcylation and phosphorylation: Roles in signaling, transcription, and chronic disease.</a> Annual Review of Biochemistry. 80 (1), 825-858 (2011).</li>
</ol>

The authors declare no competing interests.

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><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&amp;amp;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 &amp;mu;m inner diameter, 360 &amp;mu;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 H&lt;sub&gt;2&lt;/sub&gt;O)</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 &amp;micro;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 &amp;micro;L tip, 10 - 100 &amp;mu;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 &amp;mu;m, pore size 300 &amp;Aring;)</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 &amp;micro;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 ...

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Research

JoVE Journal

Biochemistry

2.2K Views.  University of Wisconsin-Madison. De dual-functionele Titanium geïmmobiliseerde metaal affiniteit chromatografie strategie kan zowel N-glycopeptiden als fosfopeptiden in dezelfde workflow verrijken, waardoor biomoleculaire informatie uit dezelfde analyse toeneemt. Het belangrijkste voordeel van deze verrijking is twee scheidingsmechanismen, die de gelijktijdige scheiding van N-glycopeptiden en fosfopeptiden in afzonderlijke fracties mogelijk maken voor downstream massaspectrometrie-analyse. Het gebruik van deze verrijkingsme...

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

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Cancer Research

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.

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 ...

Chemistry

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 ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Enhancing EV Isolation for Precise Biofluid Analysis

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

Author Spotlight: Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery 

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...

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 ...

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.

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Biology

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...

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

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