Method Article

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

DOI:

10.3791/63735

⸱

May 4th, 2022

 ,  , 
1Department of Chemistry, University of Wisconsin-Madison, 2School of Pharmacy, University of Wisconsin-Madison

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.

Pancreas tissue analysis process: cryo-pulverization, LC-MS, peptide enrichment, data analysis diagram.
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. Please click here to view a larger version of this figure.

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.

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Consent was obtained for the use of pancreatic tissues for research from the deceased'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

  1. 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.
  2. Transfer the frozen tissue pieces into the pre-chilled sample holder and add a spoonful of liquid nitrogen to the tissue.
  3. 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.
  4. 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.
  5. 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 µL 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.
  6. Add 600 µL of lysis buffer per 100 mg tissue to the tube and incubate at 95 °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.
  7. 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 °C.
  8. Add the supernatant to a 5x precipitation solvent, 300 µL 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 °C.
  9. Pellet the samples again at 3,000 x g for 15 min at 4 °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.
  10. Pellet the sample at 16,000 x g for 15 min at 4 °C. Air dry the sample pellet in a fume hood for 15 min and store at -80 °C until ready to proceed.

2. Protein digestion and desalting

  1. Resuspend the protein pellet in 300 µL of freshly made digestion buffer containing 50 mM triethylammonium bicarbonate (TEAB) and 8 M urea.
  2. Estimate the protein concentration of the solution using a protein assay according to the manufacturer's protocol.
  3. 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.
  4. Add LysC/trypsin at 1:100 enzyme:protein ratio and incubate at 37 °C for 4 h. Then, add 50 mM TEAB to dilute the 8 M urea used to < 1 M. Add trypsin at 1:100 enzyme:protein ratio and incubate at 37 °C overnight.
  5. 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.
  6. 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 > one drop per second.
  7. Elute peptides using 1 mL of 60% ACN and 0.1% formic acid (FA) solution. Dry eluted peptides at approximately 35 °C using a centrifugal vacuum concentrator until the solvent has completely evaporated.
  8. Resuspend peptides in 300 µL of water and estimate peptide concentrations using a peptide assay according to the manufacturer's protocol. Portion the peptides into 500 µg aliquots and dry completely.

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.

  1. Weigh approximately 3 mg of cotton wool and pack it into an empty 200 µL pipette tip (spin-tip; see Table of Materials).
  2. Transfer strong anion-exchange enrichment material into a tube and add 200 µL of 0.1% TFA per 10 mg material. For enrichment of 500 µg of peptides, use 15 mg of the material. Activate the material by shaking for 15 min.
  3. 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.
  4. Condition the material by centrifuging 3x with 200 µL of ACN. Repeat the triplicate conditioning using 100 mM ammonium acetate (NH4Ac), 1% TFA, and 80% ACN/0.1% TFA (loading buffer).
  5. Resuspend 500 µg peptide samples in approximately 200 µL of loading buffer and flow through the spin-tip. Reload the flow-through 2x to ensure complete binding.
  6. Wash the material by centrifuging 5x using 200 µL of 80% ACN/0.1% TFA, and then 2x using 200 µL of 80% ACN/0.1% FA.
  7. Elute the peptides by centrifuging with 200 µL 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).
  8. Wash the material by centrifuging 2x with 200 µL of 80% ACN/5% NH4OH. Elute the remaining peptides with 200 µL of 10% NH4OH (E5).
  9. Dry all the elutions completely using a centrifugal vacuum concentrator at approximately 35 °C.
  10. Perform desalting of the basic elution (E5) using a desalting tip according to manufacturer's protocol.
  11. Dry the elution from the desalting tip using a centrifugal vacuum concentrator at approximately 35 °C to completeness.

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.

  1. Weigh approximately 3 mg of cotton wool and pack it into an empty 200 µL pipette tip (spin-tip).
  2. Transfer Ti-IMAC phosphopeptide enrichment material into a tube and add 200 µL of 0.1% TFA per 10 mg material. For enrichment of 500 µg peptides, use 10 mg of material.
  3. 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.
  4. Condition the material with 200 µL of 40% ACN/3% TFA using the same centrifuge settings as described above. Resuspend peptide samples in 200 µL of 40% ACN/3% TFA and flow through the spin-tip. Reload the flow-through twice to ensure more complete binding.
  5. Wash the material by centrifuging with 200 µL of 50% ACN, 6% TFA, and 200 mM NaCl solution, followed by washing 2x in 200 µL of 30% ACN and 0.1% TFA solution.
  6. Elute peptides with 200 µL of 10% NH4OH. Dry the elution completely under vacuum. Perform desalting using a desalting tip according to the manufacturer's protocol. Dry the elution from the desalting tip under vacuum to completeness.

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.

  1. Weigh approximately 3 mg of cotton wool and pack it into an empty spin-tip.
  2. 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 µL (material can be stored in this suspension at 4 °C).
  3. For enrichment from 500 µg peptides, add enough slurry to a spin-tip to transfer 20 mg of material. Wash the spin-tip by centrifuging with 200 µL of 0.1% TFA.
  4. Resuspend the samples in 200 µL of loading/washing solvent (80% ACN and 3% TFA) and flow through the spin-tip. Reload the flow-through 2x.
  5. Wash the spin-tip 6x by centrifuging with 200 µL of loading/washing solvent. Wash the spin-tip with 200 µL of 80% ACN/0.1% FA solution.
  6. Elute the peptides with 200 µL of each of the following: 60% ACN/0.1% FA (E1) and 40% ACN/0.1% FA (E2). Analyze each elution separately.
  7. Elute the peptides with 200 µL of each of the following: 20% ACN/0.1% FA and 0.1% FA. Combine the two elutions and analyze as one sample (E3).
  8. Elute the peptides with 200 µL 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.
  9. Condition the material to basic pH using 200 µL of 90% ACN/2.5% NH4OH for 3x. Discard these washes.
  10. Elute the peptides with 200 µL 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.
  11. Elute the peptides with 200 µL 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.
  12. Dry all the elutions completely under vacuum.

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.

  1. Pull and pack a 15 cm long capillary (75 µm 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).
  2. Reconstitute enriched peptide samples in 15 µL of 3% mobile phase B. Load 2 µL of the sample (~13% sample volume) directly onto the column. Analyze each sample in technical duplicate.
  3. Elute peptides using a gradient from 3% to 30% mobile phase B over 90 min at a flow rate of 0.3 µL/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.
  4. 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.

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.

  1. Process raw data files using the appropriate software (see Table of Materials).
    1. 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.
  2. 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.
    1. 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).
    2. Set N-glycosylation as a common modification. Set the maximum total modifications per peptide spectral match (PSM) as one common and two rare.
    3. 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 > 150, |log prob| > 1, and delta mod score > 10.
    4. Export and analyze search results in the Proteins and Peptide Group tabs.
    5. 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.
  3. 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.
    1. 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.
    2. 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.
  4. Determine the number of identifications of PTMs at the protein, peptide, and modification site levels.

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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...

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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...

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors declare no competing interests.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Acetic Acid, Glacial (Certified ACS)Fisher ScientificA38S-500
Acetone (Certified ACS)Fisher ScientificA18-1
Acetonitrile, Optima LC/MS GradeFisher ScientificA955-4
Ammonium Acetate (Crystalline/Certified ACS)Fisher ScientificA637-500
Ammonium Hydroxide (Certified ACS Plus)Fisher ScientificA669-212
Byonic softwareProtein Metricsn/aCommercial software used for glycoproteomic analysis (https://proteinmetrics.com/byos/)
C18 BEH materialWaters186002353Material removed from column and used to pack nano capillaries (pulledto integrate tip used directly in line with instrument inlet)
CAE-Ti-IMAC, 100%J&K Scientific2749380-1GMaterial used for dual-functional Ti(IV)-IMAC; can also be used for conventional IMAC/conventional phosphopeptide enrichment
Cellcrusher kitCellcrushern/aUsed for grinding tissue samples into powder before extraction
Eppendorf 5424R MicrocentrifugeFisher Scientific05-401-205For temperature-controlled centrifugation
cOmplete protease inhibitor cocktail tabletsSigma11697498001
DTT, Molecular Grade (DL-Dithiothreitol)PromegaV3151Protein reducing agent
Ethanol, 200 proof (100%), USPFisher22-032-601
Fisherbrand Analog Vortex MixerFisher Scientific02-215-414
Fisherbrand Low-Retention Microcentrifuge Tubes (1.5 mL)Fisher Scientific02-681-320
Fisherbrand Low-Retention Microcentrifuge Tubes (2 mL)Fisher Scientific02-681-321
Fisherbrand Model 120 Sonic DismembratorFisher ScientificFB120110For sample lysis using ultrasonication
Formic Acid, 99.0+%, Optima LC/MS GradeFisher ScientificA117-50
Fused silica capillary (75 μm inner diameter, 360 μm outer diameter)Polymicro Technologies LLC100 m TSP075375For in-house pulled and packed columns with integrated emitter
Hydrofluoric acid (48 wt. % in H2O)Sigma-Aldrich339261-100MLUsed for opening emitter of pulled capillary column
Iodoacetamide, BioUltraSigmaI1149-5GProtein reducing reagent
MaxQuant softwaren/an/aFree software used for phosphoproteomic analysis (https://www.maxquant.org/)
Multi-therm Shaker with heating and coolingBenchmark ScientificH5000-HCHeating block
Oasis HLB 1 cc Vac Cartridge, 10 mg Sorbent per Cartridge, 30 µm, 100/pkWaters186000383Larger-scale cartridge desalting for tryptic digests (loading capacity approximately up to 1 mg each)
OMIX C18 pipette tips, 100 µL tip, 10 - 100 μL elution volume, 1 x 96 tipsAgilentA57003100Smaller-scale packed pipette tip for desalting for enrichment elutions
P-2000 Micropipette PullerSutter Instrument Co.P-2000/FFor pulling nano-capillary columns for LC-MS
PhosSTOP phosphatase inhibitor tabletsSigma4906845001
Pierce BCA Protein Assay KitThermo Fisher Scientific23225
Pierce Quantitative Colorimetric Peptide AssayThermo Fisher Scientific23275
PolySAX LP (12 μm, pore size 300 Å)PolyLCBMSX1203Material for strong anion-exchange chromatography used for ERLIC/conventional glycopeptide enrichment
Potassium Phosphate Monobasic (Crystalline/Certified ACS)Fisher ScientificP285-500
Pressure injection cell with integrated magnetic stirplateNext AdvancePC77-MAGFor packing nano-capillary columns with stationary phase up to 2500 psi limit
Proteome Discoverer softwareThermo Fisher Scientificn/aCommercial 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)
SpeedVac SC110 Vacuum Concentrator Model SC110-120Savantn/aCentrifugal vacuum concentrator for drying samples (under heat)
SDS Solution, 10% Sodium Dodecyl Sulfate Solution, Molecular Biology/ElectrophoresisFisher ScientificBP2436200
Sequencing Grade Modified TrypsinPromegaV5111
Sodium Chloride (Crystalline/Certified ACS)Fisher ScientificS271-500
TopTip, Empty, 10-200 µL, Pack of 96Glygen CorporationTT2EMT.96Empty pipette tip with micron-sized hole used that can be used to pack chromatographic materials for enrichments, bundled with tube adapters
Triethylammonium bicarbonate buffer (TEAB, 1 M, pH 8.5 (volatile))Sigma90360-100ML
Trifluoroacetic acid, Reagent Grade, 99%Fisher Scientific60-017-61
Tris Base (White Crystals or Crystalline Powder/Molecular Biology)Fisher ScientificBP152-500
Trypsin/Lys-C Mix, Mass Spec GradePromegaV5071
Urea (Certified ACS)Fisher ScientificU15-500
Water, Optima LC/MS GradeFisher ScientificW64

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Smith, L. M., Kelleher, N. L. Proteoform: a single term describing protein complexity. Nature Methods. 10 (3), 186-187 (2013).
  2. Pan, S., Brentnall, T. A., Chen, R. Glycoproteins and glycoproteomics in pancreatic cancer. World Journal of Gastroenterology. 22 (42), 9288-9299 (2016).
  3. Tabang, D. N., Ford, M., Li, L. Recent advances in mass spectrometry-based glycomic and glycoproteomic studies of pancreatic diseases. Frontiers in Chemistry. 9, 707387(2021).
  4. Khoury, G. A., Baliban, R. C., Floudas, C. A. Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database. Scientific Reports. 1, 90(2011).
  5. Alpert, A. J. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. Journal of Chromatography A. 499, 177-196 (1990).
  6. Riley, N. M., Bertozzi, C. R., Pitteri, S. J. A pragmatic guide to enrichment strategies for mass spectrometry-based glycoproteomics. Molecular & Cellular Proteomics. 20, 100029(2020).
  7. Low, T. Y., et al. Widening the bottleneck of phosphoproteomics: Evolving strategies for phosphopeptide enrichment. Mass Spectrometry Reviews. 40 (4), 309-333 (2021).
  8. Cho, K. C., Chen, L., Hu, Y., Schnaubelt, M., Zhang, H. Developing workflow for simultaneous analyses of phosphopeptides and glycopeptides. ACS Chemical Biology. 14 (1), 58-66 (2019).
  9. Zhou, Y., et al. An integrated workflow for global, glyco-, and phospho-proteomic analysis of tumor tissues. Analytical Chemistry. 92 (2), 1842-1849 (2020).
  10. Tang, R., et al. Facile preparation of bifunctional adsorbents for efficiently enriching N-glycopeptides and phosphopeptides. Analytica Chimica Acta. 1144, 111-120 (2021).
  11. Wang, Z., Wang, J., Sun, N., Deng, C. A promising nanoprobe based on hydrophilic interaction liquid chromatography and immobilized metal affinity chromatography for capture of glycopeptides and phosphopeptides. Analytica Chimica Acta. 1067, 1-10 (2019).
  12. Glover, M. S., et al. Characterization of intact sialylated glycopeptides and phosphorylated glycopeptides from IMAC enriched samples by EThcD fragmentation: Toward combining phosphoproteomics and glycoproteomics. International Journal of Mass Spectrometry. 427, 35-42 (2018).
  13. Alpert, A. J. Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides. Analytical Chemistry. 80 (1), 62-76 (2008).
  14. Cui, Y., et al. Counterion optimization dramatically improves selectivity for phosphopeptides and glycopeptides in electrostatic repulsion-hydrophilic interaction chromatography. Analytical Chemistry. 93 (22), 7908-7916 (2021).
  15. Cui, Y., et al. Finding the sweet spot in ERLIC mobile phase for simultaneous enrichment of N-Glyco and phosphopeptides. Journal of the American Society for Mass Spectrometry. 30 (12), 2491-2501 (2019).
  16. Tabang, D. N., et al. Analysis of pancreatic extracellular matrix protein post-translational modifications via electrostatic repulsion-hydrophilic interaction chromatography coupled with mass spectrometry. Molecular Omics. 17 (5), 652-664 (2021).
  17. Huang, J., et al. Dual-functional Titanium(IV) immobilized metal affinity chromatography approach for enabling large-scale profiling of protein Mannose-6-Phosphate glycosylation and revealing its predominant substrates. Analytical Chemistry. 91 (18), 11589-11597 (2019).
  18. Huang, J., et al. Dual-functional Ti(IV)-IMAC material enables simultaneous enrichment and separation of diverse glycopeptides and phosphopeptides. Analytical Chemistry. 93 (24), 8568-8576 (2021).
  19. Jami-Alahmadi, Y., Pandey, V., Mayank, A. K., Wohlschlegel, J. A. A robust method for packing high resolution C18 RP-nano-HPLC columns. Journal of Visualized Experiments: JoVE. (171), e62380(2021).
  20. Bern, M., Kil, Y. J., Becker, C. Byonic: advanced peptide and protein identification software. Current Protocols in Bioinformatics. , Chapter 13, Unit13.20 (2012).
  21. Tyanova, S., Temu, T., Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nature Protocols. 11 (12), 2301-2319 (2016).
  22. Perez-Riverol, Y., et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Research. 47, 442-450 (2019).
  23. Zhou, Y., et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nature Communications. 10 (1), 1523(2019).
  24. Zacharias, L. G., et al. HILIC and ERLIC enrichment of glycopeptides derived from breast and brain cancer cells. Journal of Proteome Research. 15 (10), 3624-3634 (2016).
  25. Yang, W., et al. Comparison of enrichment methods for intact N- and O-linked glycopeptides using strong anion exchange and hydrophilic interaction liquid chromatography. Analytical Chemistry. 89 (21), 11193-11197 (2017).
  26. Toghi Eshghi, S., Shah, P., Yang, W., Li, X., Zhang, H. GPQuest: A spectral library matching algorithm for site-specific assignment of tandem mass spectra to intact N-glycopeptides. Analytical Chemistry. 87 (10), 5181-5188 (2015).
  27. Liu, M. -Q., et al. pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification. Nature Communications. 8 (1), 438(2017).
  28. Lu, L., Riley, N. M., Shortreed, M. R., Bertozzi, C. R., Smith, L. M. O-Pair Search with MetaMorpheus for O-glycopeptide characterization. Nature Methods. 17 (11), 1133-1138 (2020).
  29. Caval, T., Heck, A. J. R., Reiding, K. R. Meta-heterogeneity: Evaluating and describing the diversity in glycosylation between sites on the same glycoprotein. Molecular & Cellular Proteomics. 20, 100010(2021).
  30. Lee, J. S., Smith, E., Shilatifard, A. The language of histone crosstalk. Cell. 142 (5), 682-685 (2010).
  31. Leutert, M., Entwisle, S. W., Villén, J. Decoding post-translational modification crosstalk with proteomics. Molecular & Cellular Proteomics. 20, 100129(2021).
  32. Hart, G. W., Slawson, C., Ramirez-Correa, G., Lagerlof, O. Cross talk between O-GlcNAcylation and phosphorylation: Roles in signaling, transcription, and chronic disease. Annual Review of Biochemistry. 80 (1), 825-858 (2011).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

N Glycopeptide EnrichmentPhosphopeptide EnrichmentSpin Tip MethodTitanium IMACMass SpectrometryPancreatic TissuePost Translational ModificationsGlycosylation PhosphorylationProtein DigestionBiomarker Discovery

Related Articles