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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Methods Mol Biol. 2021;2259:247–257. doi: 10.1007/978-1-0716-1178-4_16

Mass spectrometry-based proteomics for analysis of hydrophilic phosphopeptides

Chia-Feng Tsai 1, Jeffrey S Smith 2,3, Dylan S Eiger 2,3, Kendall Martin 1, Tao Liu 1, Richard D Smith 1, Tujin Shi 1, Sudarshan Rajagopal 2,3, Jon M Jacobs 1,
PMCID: PMC8071202  NIHMSID: NIHMS1691540  PMID: 33687720

Abstract

Protein phosphorylation is a critical post-translational modification (PTM), with cell signaling networks being tightly regulated by protein phosphorylation. Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides that often have multiple phosphorylation sites. Herein we describe an MS-based phosphoproteomics protocol for effective quantitative analysis of hydrophilic phosphopeptides. This protocol was built upon a simple tandem mass tag (TMT)-labeling method for significantly increasing peptide hydrophobicity, thus effectively enhancing RPLC-MS analysis of hydrophilic peptides. Through phosphoproteomic analyses of MCF7 cells, this method was demonstrated to greatly increase the number of identified hydrophilic phosphopeptides and improve MS signal detection. With the TMT labeling method, we were able to identify a previously unreported phosphopeptide from the G protein-coupled receptor (GPCR) CXCR3, QPpSSSR, which is thought to be important in regulating receptor signaling. This protocol is easy to adopt and implement, and thus should have broad utility for effective RPLC-MS analysis of the hydrophilic phosphoproteome as well as other highly hydrophilic analytes.

Keywords: Hydrophilic phosphopeptide, Phosphoproteomics, TMT labeling, Phosphopeptide enrichment, Mass spectrometry

1. Introduction

Cell signaling is highly regulated by protein phosphorylation [1–3]. Changes in phosphorylation stoichiometry are used as an indicator of signaling pathway activation (cellular functional states) [1–3]. Aberrant protein phosphorylation is linked to human diseases including cancer [2–4]. Mass spectrometry (MS)-based phosphoproteomics has emerged as a powerful tool for comprehensive, site-specific, quantitative phosphoproteome profiling. Due to the low stoichiometry of protein phosphorylation and low ionization efficiency of phosphopeptides, enrichment of phosphopeptides (e.g., immobilized metal affinity chromatography (IMAC) and metal oxide chromatography (MOC)) is used for their effective MS analysis [5, 6]. The combined efficient phosphopeptide enrichment with advanced LC-MS platforms enables routine identification of more than 10,000 phosphorylation sites in a single LC-MS/MS analysis (or 50,000 sites with multi-dimensional separation approaches) [7–12]. Despite these advances, significant gaps in phosphoproteome coverage still exist, especially for small, hydrophilic phosphopeptides with low abundance. The use of trypsin, the protease of choice for most shotgun proteomics studies [13], generates peptides well suited for ESI-MS [14]. However, some peptides containing hydrophilic phosphate groups can become too hydrophilic to be retained by the widely utilized reversed-phase (RP) LC columns for separation of peptide mixtures prior to MS. Therefore, current MS-based phosphoproteomics platforms may inadvertently overlook certain portions of the highly hydrophilic phosphoproteome.

We recently developed a simple, easily implemented method to introduce a commonly used tandem mass tag (TMT) to increase peptide hydrophobicity, effectively enhancing RPLC-MS analysis of hydrophilic phosphopeptides [15]. TMT reagents are broadly used in shotgun proteomics for multiplexed sample analysis by labeling primary amines (N-terminus and ε-amine group of lysine) with chemical tags though N-hydroxysuccinimide (NHS) chemistry. Different from conventional TMT labeling, this TMT-assisted method capitalizes on TMT labeling occurring before C18-based solid phase extraction (SPE) to avoid the loss of hydrophilic phosphopeptides during C18 SPE cleanup and uses a no-primary amine buffer for sample preparation so that TMT reagents can be effectively labeled on peptides before C18 SPE (Fig. 1). In this chapter, we describe in detail a protocol for the TMT-assisted method that can be used for convenient quantitative phosphoproteome profiling of very hydrophilic phosphopeptides. We highlight critical steps in sample preparation for phosphoproteome analysis and briefly describe the analysis of phosphoproteomics data using MaxQuant [16, 17] and demonstrate how the protocol is appropriate for global analysis of the hydrophilic phosphoproteome and rescuing highly hydrophilic phosphopeptides with important biological functions.

Figure 1.

Figure 1.

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Workflow for MS-based phosphoproteomic analysis of cell lysate digests (left: standard method; right: TMT-based method).

We systematically evaluated the effect of the TMT-based method on global phosphoproteomics analysis. Using equal amounts of tryptic peptides from MCF-7 cell lysates with or without TMT0 labeling, we demonstrated that the number of identified phosphopeptides with TMT0 labeling (N=10,311) was comparable to that without TMT0 labeling (N=11,256) (Figure 2a). A total of 16,104 unique phosphopeptides were identified from the two sets of samples with 5,463 (34%) phosphopeptides in common (Figure 2b), an overlap that is significantly lower than the typical overlap for replicate analysis of phosphopeptides (~50%), suggesting that TMT labeling allowed for the identification of phosphopeptides that otherwise would have been overlooked with traditional analytical strategies. Indeed, evaluation of the changes in peptide retention time before and after TMT0 labeling showed that TMT0 labeling led to greatly increased peptide retention time (Figure 2c), and thus allowed the identification of phosphopeptides with lower hydrophobicity (Figure 2d). We further used the TMT-based method to identify a previously unreported phosphopeptide located on the C-terminus of the GPCR CXCR3, QPpSSSR, that is thought to contribute to the regulation of its signaling mechanism. This simple method is expected to be broadly used not only for profiling hydrophilic phosphoproteomes, but also for effective RPLC-MS analysis of other highly hydrophilic analytes.

Figure 2.

Figure 2.

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Phosphoproteomic analysis of MCF7 cell lysate digests without and with TMT0 labeling. (a) The number of identified phosphopeptides with and without TMT labeling. (b) the overlap of identified phosphopeptides between with and without TMT labeling. (c) The median of retention time shift of phosphopeptides with versus without TMT labeling. (d) The hydrophobicity distribution of identified phosphopeptides.

2. Materials

All buffers are made with sequencing grade chemicals and ultrapure water (Milli-Q).

2.1. Cell Lysis

Experiments were performed using MCF7 and HEK 293 cell lines in ATCC-formulated Eagle’s minimum essential medium supplemented with 0.01 mg/mL human recombinant insulin and a final concentration of 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific). Use appropriate media for your cell line of choice.

  1. ATCC MCF7 and HEK 293 cell lines.

  2. Sterile petri dishes (150 mm).

  3. Phosphatase inhibitor cocktail.

  4. Lysis buffer (MCF7): 100 mM NH4HCO3, pH 8.0, 8 M urea, and a 1% phosphatase inhibitor.

  5. Lysis buffer (HEK 293): 50 mM Tris HCl, 150 mM NaCl, 1% NP-40 at pH 7.5 containing a phosphatase inhibitor.

  6. Phosphate-buffered saline (PBS).

  7. Rubber policeman cell scraper.

  8. Sonicator UTR200 for cell lysis.

  9. Eppendorf centrifuge 5810R for centrifugation.

  10. BCA-based protein concentration quantification assay.

2.2. Protein Digestion

  1. 200 mM dithiothreitol (DTT).

  2. 300 mM fresh iodoacetamide (IAA).

  3. 1 M trimethyl ammonium bicarbonate (TMAB).

  4. Sequencing grade modified trypsin (Promega).

  5. Trifluoroacetic acid (TFA).

  6. SpeedVac concentrator.

2.3. TMT Labeling

  1. Reconstitution buffer: 100% ACN.

  2. Reaction buffer: 200 mM HEPES (pH 8.5).

  3. TMT0 reagent.

  4. Stopping reagent: 5% hydroxylamine.

  5. Acidifying reagent: 10% TFA.

2.4. Peptide Desalting

  1. Reversed phase Waters C18 Sep-Pak cartridge.

  2. 3M Empore C18 membrane disks.

  3. 16G and 20G flat-bottom needle and plunger.

  4. 200 μL tip for low peptide input.

  5. Wash buffer: 100% methanol (MeOH).

  6. Buffer A: 0.1% TFA.

  7. Buffer B: 80% ACN in 0.1% TFA.

  8. SpeedVac concentrator.

2.5. Phosphopeptide Enrichment

  1. Ni-NTA silica resin.

  2. Self-pack IMAC tip.

  3. 20 μm polypropylene frit disk.

  4. 18G flat-bottom needle and plunger.

  5. 50 mM EDTA in 1 M NaCl.

  6. Equilibration buffer (IMAC): 1% acetic acid.

  7. Activation buffer: 100 mM FeCl3 in equilibration buffer.

  8. Loading buffer (IMAC): 80%ACN in 0.1%TFA.

  9. Wash buffer (IMAC): 80% ACN in 1% TFA.

  10. Elution buffer (IMAC): 200 mM phosphate salt.

  11. 3M Empore C18 membrane disks.

  12. C18 StageTip.

  13. Wash buffer (C18): 0.1% FA.

  14. Elution buffer (C18): 80% ACN/0.1%TFA.

  15. SpeedVac concentrator.

2.6. Liquid Chromatography and Electrospray Tandem Mass Spectrometry

  1. Phosphopeptide reconstitution buffer: 0.1%FA with 2% ACN.

  2. A nanoACQUITY UPLC system.

  3. An in-house packed analytical column (75 μm i.d. × 20 cm) containing 1.9-μm ReproSil C18 resin with a column heater set at 50 °C.

  4. Buffer A (RPLC): 0.1% FA with 3%ACN.

  5. Buffer B (RPLC): 0.1% FA in 90% ACN.

  6. Orbitrap Fusion Lumos Tribrid mass spectrometer.

  7. Data analysis software using MaxQuant software (www.maxquant.org).

3. Methods

3.1. Cell Lysis

  1. Gently aspirate cell culture media and wash cells twice with ice cold PBS

  2. Add lysis buffer to the MCF7 and HEK 293 cells.

  3. Heat solution for 5 mins at 95 °C to facilitate lysis and to inactivate endogenous proteases and phosphatases.

  4. Homogenize the lysate with sonication at 4 °C.

  5. Perform a BCA assay to determine the protein concentration.

  6. Dilute all samples to equal protein concentrations based on BCA assay results to achieve the desired starting concentration (e.g., 1–10 μg/μL).

3.2. Protein Digestion

  1. Reduce disulfide bonds using 5 mM DTT for 1 h at 37 °C.

  2. Alkylate cysteine residues using 20 mM iodoacetamide in the dark for 1 h at room temperature.

  3. Digest with trypsin at an enzyme-to-substrate ratio of 1:50 (w/w) and incubate overnight at 37 °C with gentle shaking.

  4. Acidify the tryptic peptides with TFA at a final concentration of 0.5% (v/v).

3.3. Digestion of immunoprecipitated proteins (Phosphorylated CXCR3 in this example)

  1. Treat HEK293 cells with and without the CXCR3 ligand CXCL10 at a final concentration of 100 nM for 5 mins.

  2. Lyse cells as described in 3.1.

  3. Incubate cell lysates with anti-FLAG magnetic beads for at least 4 hours at 4°C.

  4. Elute FLAG-CXCR3 with 0.1M Glycine HCl buffer (pH 3.0) followed by neutralization with 0.5 M Tris HCl, pH 7.4, 1.5 M NaCl.

  5. Precipitate proteins with 100% acetone and then air-dry.

  6. Reconstitute the immunoprecipitated CXCR3 with 200 mM HEPES (pH=8.5) and directly digest with sequencing grade modified trypsin overnight at 37 °C.

  7. Label the digested peptides directly with TMT0 reagents (see Note 1).

3.4. TMTzero (TMT0) Labeling

  1. Reconstitute the tryptic peptides with 200 mM HEPES (pH 8.5).

  2. Check the pH value (see Note 2)

  3. Mix the tryptic peptides with a TMT0 reagent using the recently optimized TMT-to-peptide ratio of 1:1 (w/w) (see Note 3).

  4. Incubate for 1 h at room temperature and terminate the reaction by the addition of 5% hydroxylamine for 15 mins.

  5. Acidify the TMT0-labeled peptides using 10%TFA with the final concentration of 0.5% TFA.

  6. Dilute the TMT0-labeled peptides with buffer A (RPLC) (see Note 4).

  7. Desalt the TMT0-labeled peptides using a C18 SPE cartridge (see 3.5) for large amounts of peptides or a C18 StageTip for small amounts of peptides (≤20 μg) (see 3.7).

3.5. Peptide Desalting by C18 Cartridge for large amounts of samples

  1. Activate a Waters Sep-Pak C18 cartridge with 1 mL MeOH and then buffer B.

  2. Equilibrate the C18 cartridge with 1 mL buffer A.

  3. Load the tryptic peptides onto the C18 cartridge.

  4. Wash the C18 cartridge with 1 mL buffer A twice.

  5. Elute the peptides with 500 μL buffer B.

  6. Determine the amount of tryptic peptides by BCA and then dry using a SpeedVac.

3.6. Phosphopeptide Enrichment by IMAC

  1. Prepare IMAC tip (20 μL pipette tip) by capping one end with a 20 μm polypropylene frits (see Note 5).

  2. Resuspend 20 mg of Ni-NTA silica resin from Ni-NTA spin column with 200 μL of 1% acetic acid (see Note 6).

  3. Transfer the resuspended Ni-NTA silica resin to the IMAC tip and then pack the Ni-NTA beads by centrifugation at 1,000 g at 25 °C for 1 min (see Note 7).

  4. Remove Ni2+ ions with 200 μL of 50 mM EDTA in 1 M NaCl.

  5. Wash away the remining 50 mM EDTA with 200 μL of 1% acetic acid (see Note 8).

  6. Activate the IMAC tip with 200 μL of 100 mM FeCl3 by centrifugation at 300 g at 25 °C for 2 mins.

  7. Equilibrate the IMAC tip with 200 μL of 1% acetic acid before sample loading.

  8. Reconstitute the desalted TMT0-lableled peptides with 100 μL 80%ACN 0.1%TFA (see Note 9).

  9. Load the desalted peptides onto the prepared IMAC tip by centrifugation at 300 g at 25 °C for 2 mins.

  10. Wash the IMAC tip with 100 μL 80%ACN 0.1%TFA to remove unbound nonphosphopeptides by centrifugation at 300 g at 25 °C for 2 mins (see Note 10).

  11. Wash the IMAC tip with 100 μL 80%ACN 1%TFA to remove unbound nonphosphopeptides by centrifugation at 300 g at 25 °C for 2 mins (see Note 10).

  12. Wash the IMAC tip with 100 μL 1% acetic acid to remove unbound nonphosphopeptides by centrifugation at 300 g at 25 °C for 2 mins (see Note 11).

  13. Elute the bound phosphopeptides with 200 μL of 200 mM NH4H2PO4 by centrifugation at 200 g at 25 °C for 2 mins. Repeat this step one more time.

  14. Transfer the eluted phosphopeptides to the C18 StageTip by centrifugation at 1,000 g at 25 °C for 4 mins.

3.7. Peptide Desalting by C18 StageTip for small amounts of samples

  1. Use a 16 Gauge needle to punch a hole on the C18 membrane disk.

  2. Use a 20 Gauge needle to help insert the punched membrane disk into the D200 tip (the loading capacity of ~20 μg) (see Note 12).

  3. Activate the C18 StageTip with 40 μL MeOH and then buffer B.

  4. Equilibrate the C18 StageTip with 40 μL buffer A.

  5. Load the enriched phosphopeptides onto the C18 StageTip.

  6. Wash the C18 StageTip with 40 μL buffer A for two times.

  7. Elute phosphopeptides directly into the sample vial with 40 μL buffer B.

  8. Dry down the phosphopeptides with SpeedVac and then store at −80 °C until LC-MS analysis.

  9. Reconstitute the phosphopeptides with 2%ACN 0.1% FA for LC-MS analysis.

3.8. LC-MS Setup

Example with Waters nanoACQUITY LC and Orbitrap Fusion Lumos Tribrid mass spectrometer.

A column was packed in-house using 20 cm long fused silica with 75 μm inner diameter and packed with 1.9-μm ReproSil C18 resin.

  1. Load 5 μL of sample (reconstituted in 4%ACN 0.1%FA) on the column and separate through a gradient (2–6% solvent B in 1 min, 6–30% solvent B in 84 min, 30–60% solvent B in 9 min, 60–90% solvent B in 1 min, and finally 90% solvent B for 5 min) at a flow rate of 200 nL/min.

  2. Run the Lumos Tribrid mass spectrometer in the data dependent mode at a top 12 method. Resolution for full scans 60,000, the AGC value of 4 × 105, maximum injection time of 50 ms, and scan range from 300–1500 m/z. Resolution for HCD MS/MS scan 50,000, the AGC value of 1 × 105, maximum injection time of 300 ms, a normalized collision energy of 30%, a cycle time of 2 s, and a dynamic exclusion time of 45 s.

3.9. Data analysis

  1. Analyze the raw files using the freely available MaxQuant against human UniProt database (version May 20, 2015). Variable modifications: the acetylation (protein N-term), oxidation (M), and phospho (STY); the fixed modification: the carbamidomethyl (C). 1% false discovery rate (FDR) is used for the levels of protein, peptide, and modification.

  2. Results can be read out from the modificationSpecificPeptides and Phospho (STY) Sites tab-delimited txt output files generated by MaxQuant.

4. Notes

  1. QPSSSR and its phosphorylated forms from CXCR3 are too hydrophilic to detect using standard protocols, as without TMT0 labeling they cannot bind to a C18 column or cartridge.

  2. The TMT labeling efficiency is sensitive to the buffer pH. Prior to the TMT0 labeling we test the pH value to ensure pH≥8 using pH paper. This is a key step for high-efficiency TMT labeling.

  3. We employ the recently optimized TMT-to-peptide ratio of 1:1 (w/w) to have both robust and cost-efficient TMT0-labeing [18].

  4. After TMT0 labeling the ACN concentration need to be reduced below 5%. The remaining high concentration of ACN will affect the recovery of TMT0-labeled peptides for C18 desalting to remove unreacted TMT0 reagent.

  5. For preparation of IMAC tip, the frits are soft and seals very easily at the end of tip. Applying a large pressure results in more tightly packing and the back pressure will be too high to be difficult for buffer passing through the IMAC tip.

  6. 2 mg of Ni-NTA silica resin is used for ≤100 μg of tryptic peptides. Adjustment is required for small amounts of tryptic peptides.

  7. Ni-NTA silica resin settles very quickly and must be mixed thoroughly immediately before pipetting to ensure that the resin is evenly distributed

  8. It is critical to wash away the remaining 50 mM EDTA for phosphopeptide binding to IMAC tip.

  9. The IMAC loading buffer must be freshly prepared because of high concentration of ACN. The ACN concentration will affect the specificity of phosphopeptide enrichment.

  10. The IMAC wash buffer must be freshly prepared because of high concentration of ACN. The ACN concentration will affect the specificity of phosphopeptide enrichment.

  11. It is critical to wash away the remaining ACN in the IMAC tip because it will affect the recovery of phosphopeptides for C18 desalting.

  12. For C18 StageTip, the membrane disk is soft and seals very easily at its sides. Applying a larger pressure at this point results in a more densely packed disk and raises the back pressure of the StageTip unnecessarily.

Acknowledgements

Portions of the research were supported by P41GM103493 (R.D.S), R21CA223715 (T.S.), T32GM7171 (J.S.S.), the Duke Medical Scientist Training Program (J.S.S.), R01GM122798 (S.R.), and Burroughs Wellcome Career Award for Medical Scientists (S.R.).

The experimental work described herein was performed in the Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, a national scientific user facility sponsored by the United States of America Department of Energy under Contract DE-AC05-76RL0 1830.

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