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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Methods Mol Biol. 2021;2262:47–64. doi: 10.1007/978-1-0716-1190-6_3

Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry

Lauren M Adams 1, Caroline J DeHart 2, Neil L Kelleher 1,3,4
PMCID: PMC8543976  NIHMSID: NIHMS1745279  PMID: 33977470

Abstract

The characterization of biologically relevant post-translational modifications (PTMs) on KRAS4B has historically been carried out through methodologies such as immunoblotting with PTM-specific antibodies or peptide-based proteomic methods. While these methods have the potential to identify a given PTM on KRAS4B, they are incapable of characterizing or distinguishing the different molecular forms or proteoforms of KRAS4B from those of related RAS isoforms. We present a method that combines immunoprecipitation of KRAS4B with top-down mass spectrometry (IP-TDMS), thus enabling the precise characterization of intact KRAS4B proteoforms. We provide detailed protocols for the IP, LC-MS/MS, and data analysis comprising a successful IP-TDMS assay in the contexts of cancer cell lines and tissue samples.

Keywords: RAS, Isoform, Proteoform, Top-down mass spectrometry, Immunoprecipitation, Intact protein

1. Introduction

The four RAS isoforms, KRAS4A, KRAS4B, HRAS, and NRAS, share up to 90% sequence identity within the first 165 residues. The sequences diverge at the C-terminal hypervariable regions of each isoform, which contain PTMs important for proper membrane association, protein-protein interactions, and downstream signaling cascades [1-5]. Mass spectrometry is an important tool for the identification and localization of PTMs on endogenous proteins, including those involved in RAS-dependent signaling pathways. Historically, mass spectrometry-based proteomic studies have employed proteolytic digestion using enzymes such as trypsin and subsequent analysis of the resulting peptides, in what is termed the “bottom-up” (BU) approach [6]. Due to the high sequence identity within the RAS protein family, a BU approach to identify and localize RAS PTMs results in the generation of multiple peptides with the same sequence, despite originating from different RAS isoforms or mutational variants. Additionally, with the high density of C-terminal lysines and the labile nature of key PTMs, it has proven impossible to precisely and comprehensively characterize RAS proteoforms by BU [7]. Alternatively, top-down mass spectrometry (TDMS) bypasses proteolytic digestion and measures the intact mass of proteins. While TDMS offers a way to precisely characterize RAS proteoforms in which BU does not, the TDMS workflow presents a few technical challenges (Fig. 1) [6, 7]. Foremost among these is the requirement for a certain starting concentration of target proteoforms in order to exceed the limit of detection for the instrument, thus permitting successful accurate mass determination and confident sequence characterization or PTM localization [8]. This limitation can be overcome by enriching the target proteoform population(s) prior to TDMS analysis by methods such as immunoprecipitation (IP). A study in 2018 by Ntai et al. developed a KRAS4B assay entailing IP and subsequent TDMS analysis (IP-TDMS), which was then successfully implemented to compare endogenous KRAS4B proteoforms between different mutational contexts in colorectal cancer cell lines and patient tumor samples [7].

Fig. 1.

Fig. 1

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Graphical representation depicting three modes of proteomic analysis: SDS-PAGE (e.g., western blot, silver nitrate stain), bottom-up proteomics, and top-down proteomics. As experimental difficulty increases, so does the quantity of information obtained by characterization of the target protein(s)

Analysis of KRAS4B proteoforms by IP-TDMS provides a largely unbiased readout of PTMs with stoichiometry preserved and an improved view of global KRAS4B proteoform differences when comparing between cancer cell line or tumor contexts. Moreover, IP-TDMS of KRAS4B offers precise biochemical knowledge to make the assignment of PTM function more efficient in basic and translational research. Herein, we describe in detail the standardized protocol (SOP) used for KRAS4B IP-TDMS first published in Ntai et al. (2018) [7], which employs commercially available α-v- HRAS IP beads and is the most robust method for characterizing KRAS4B proteoforms within tissue samples embedded in optimal cutting temperature (OCT) cryopreservation medium [7]. We also present an alternative IP-TDMS method employing magnetic IP beads, thus allowing for antibody-specific customization of the IP workflow. Both IP-TDMS protocols present researchers with a method of examining KRAS4B proteoform populations in the context of cancer to a degree inaccessible using BU approaches [6].

2. Materials

2.1. SOP for KRAS4B Immunoprecipitation

  1. IP beads: Millipore α-v-H-RAS (Y13–259) agarose conjugate (OP01A-0.5ML).

  2. Ice bath.

  3. 15 and 50 mL conical tubes.

  4. Cryomill (Retsch Mixer Mill MM400 or similar, with accompanying 5 or 25 mL grinding jars) for cryopreserved tissue samples.

  5. Tip sonicator.

  6. Lysis buffer: 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40 (IGEPAL CA-360), 1 × HALT Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific).

  7. Wash buffer 1: 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40 (IGEPAL CA-360), 1 × HALT Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific).

  8. Wash buffer 2: 50 mM Tris (pH 7.5), 150 mM NaCl.

  9. Elution buffer: 0.5% MS-grade trifluoroacetic acid (TFA) in Optima-grade H2O (Thermo Fisher Scientific).

  10. Centrifuge (e.g., Eppendorf 5804R 15 amp version, refrigerated, swinging bucket rotor S-4–72).

  11. Microcentrifuge (e.g., Fisher Scientific accuSpin Micro 17, 75003524, Ch. 500010PP).

  12. Tube rotator.

  13. ThermoMixer (e.g., Eppendorf, 24 × 1.5 mL block).

  14. 4 °C cold room or refrigerator.

  15. C4 ZipTips (Millipore Sigma).

  16. C4 ZipTip activation buffer: 100% Optima-grade acetonitrile (ACN, Thermo Fisher Scientific).

  17. C4 ZipTip wash buffer: 0.2% MS-grade formic acid (FA) in Optima-grade H2O.

  18. C4 ZipTip elution buffer: 0.2% MS-grade FA, 80% Optima- grade ACN in Optima-grade H2O.

  19. HPLC Solvent A: 0.2% MS-grade FA, 5% Optima-grade ACN in Optima-grade H2O.

  20. 1.5 mL Protein LoBind Tubes (Eppendorf).

  21. 2 mg/mL bovine serum albumin (BSA).

  22. Autosampler vials.

2.2. KRAS4B Magnetic Bead Immunoprecipitation

  1. Protein A/G magnetic beads (or magnetic beads with an alternative ligand type compatible with the antibody of choice).

  2. α-v-HRAS (Y13–259) antibody (or alternative antibody of choice).

  3. Magnetic tube rack or other strong magnet.

  4. 1 × Tris-buffered saline (TBS): 50 mM Tris (pH 7.5), 150 mM NaCl.

  5. Ice bath.

  6. 15 and 50 mL conical tubes.

  7. Cryomill (Retsch Mixer Mill MM400 or similar, with accompanying 5 or 25 mL grinding jars) for cryopreserved tissue samples.

  8. Tip sonicator.

  9. Lysis buffer: 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40 (IGEPAL CA-360), 1 × HALT Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific).

  10. Wash buffer 1: 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40 (IGEPAL CA-360), 1 × HALT Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific).

  11. Wash buffer 2: 50 mM Tris (pH 7.5), 150 mM NaCl.

  12. Elution buffer: 0.5% MS-grade trifluoroacetic acid (TFA, Milli- pore) in Optima-grade H2O.

  13. Centrifuge (e.g., Eppendorf 5804R 15 amp version, refrigerated, swinging bucket rotor S-4–72).

  14. Microcentrifuge (e.g., Fisher Scientific accuSpin Micro 17, 75003524, Ch. 500010PP).

  15. Tube rotator.

  16. ThermoMixer (e.g., Eppendorf, 24 × 1.5 mL block).

  17. 4 °C cold room or refrigerator.

  18. C4 ZipTips (Millipore Sigma).

  19. C4 ZipTip activation buffer: 100% Optima-grade acetonitrile (ACN).

  20. C4 ZipTip wash buffer: 0.2% MS-grade FA in Optima-grade H2O.

  21. C4 ZipTip elution buffer: 0.2% MS-grade FA, 80% Optima- grade ACN in Optima-grade H2O.

  22. HPLC Solvent A: 0.2% MS-grade FA, 5% Optima-grade ACN in Optima-grade H2O.

  23. 1.5 mL Protein LoBind Tubes (Eppendorf).

  24. 2 mg/mL bovine serum albumin (BSA).

  25. Autosampler vials.

2.3. LC-MS/MS

  1. Dionex UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific).

  2. HPLC Solvent A: 95% Optima-grade H2O, 5% Optima-grade ACN, 0.2% FA.

  3. HPLC Solvent B: 5% Optima-grade H2O, 95% Optima-grade ACN, 0.2% FA.

  4. Trap column: 150 μm ID x 3 cm L, packed in-house with polymeric reverse-phase PLRP-S media (5 μm dp, 1000 A pore size, Agilent Technologies). Commercially available equivalent: PepSwift trap column, 200 μm ID × 0.5 cm L, monolithic PVDB reverse-phase media, Thermo Fisher Scientific (#164558).

  5. Nanobore analytical column: 75 μm ID × 25 cm L, packed in-house with polymeric reverse-phase PLRP-S media (5 μm dp, 1000 A pore size, Agilent Technologies). Commercially available equivalent: ProSwift RP-4H nanocapillary analytical column, 100 μm ID × 50 cm L, monolithic PVDB reverse- phase media, Thermo Fisher Scientific (#164921).

  6. Spray emitter: New Objective (FS3605015N20), 15 μm, packed in-house with polymeric reverse-phase PLRP-S media (5 μm dp, 1000 A pore size, Agilent Technologies). Commercially available equivalent: Nanospray flex source (#ES071) with stainless steel emitters (#ES542), Thermo Fisher Scientific.

  7. Q-Exactive HF BioPharma mass spectrometer (Thermo Fisher Scientific).

2.4. Data Analysis

  1. Xcalibur QualBrowser v4.0.27.10 (Thermo Fisher Scientific) with Xtract license (available to Thermo instrument owners by request from ThermoMSLicensing@thermo.com).

  2. ProSight Lite v1.4 (available for free download: http://prosightlite.northwestern.edu/) [9].

3. Methods

3.1. SOP for KRAS4B Immunoprecipitation (Fig. 2)

Fig. 2.

Fig. 2

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Experimental workflow schematic for KRAS4B IP-TDMS assays employing either agarose or custom magnetic immunoprecipitation (IP) beads (figure modified from Ntai et al.) [7]. Day 1 of the SOP workflow entails cancer cell or tissue lysis, followed by whole cell lysate incubation with IP beads overnight (see Subheadings 3.1 and 3.2). Day 2 of the SOP workflow involves completion of the IP, desalting and concentration of the immunoenriched KRAS4B protein, and proteoform identification by top-down LC-MS/ MS (see Subheadings 3.1—3.4)

Day 1

  1. Prepare lysis buffer and chill on ice. Add HALT Inhibitor Cocktail only to chilled buffer immediately prior to lysis.

  2. For cell culture (~1 × 108 cells recommended), rinse cells twice with ice-cold PBS, and store on ice. Add ~1 mL of lysis buffer per ~1 × 107 cells (for a 15 cm cell culture dish, this is approximately 2 mL per dish). For cell pellets, thaw cells on ice, followed by resuspension in 8 mL chilled lysis buffer.

  3. For cryopreserved tissue samples (>200 mg recommended), maintain in liquid nitrogen (LN2) until just before cryogrind- ing. Subject to two rounds of grinding (frequency 30 Hz, 2 min), keeping samples immersed in LN2 between rounds. Transfer pulverized lysate to a 50 mL conical tube pre-chilled in LN2. Allow lysate to thaw on ice, followed by resuspension in 25 mL chilled lysis buffer.

  4. Incubate lysate on ice for 30 min. Pipette the lysate every 10 min to ensure even resuspension. Do not vortex. If lysing on dish, use a cell scraper and serological pipette to transfer lysate to a clean conical tube.

  5. Sonicate the lysate on ice at 50% amplitude for 30 2-s cycles, with incubation on ice for 5 s in between each cycle.

  6. Clarify the lysate by centrifugation for 30 min at 16,000 x g and 4 °C.

  7. Equilibrate 50 μΕοίΙΡ beads (100 μΕοί50% slurry) in 1 mLof lysis buffer in a 15 mL conical tube on ice for 30 min. Remove the majority of lysis buffer while still leaving a visible layer of buffer on top of the beads (see Note 1).

  8. Take gel aliquots (for western blot) from the lysate supernatant, and store at —20 °C or —80 °C (see Note 2).

  9. Add lysate to the IP beads. For lysate from tissue samples (25 mL), divide evenly between two aliquots of IP beads (12.5 mL/50 μL IP beads). Incubate for 16–20 h at 4 °C with constant rotation.

  10. Prepare one autosampler vial per IP by adding 200 μL of 2 mg/mL BSA. Store vial in a closed container at 4 °C until use during Day 2 of IP (see Note 3).

Day 2

  • 11.

    Prepare IP wash buffer 1 and 2. Chill on ice.

  • 12.

    Collect lysate flow through centrifugation for 5 min at 120 x g and 4 °C (see Note 4).

  • 13.

    Take gel aliquots from the flow through supernatant and store at —20 °C or —80 °C.

  • 14.

    Remove supernatant, leaving a visible layer of liquid covering the beads (see Note 1).

  • 15.

    Wash the IP beads three times with 5 mL of wash buffer 1. Pellet beads by centrifugation for 5 min at 120 x g and 4 °C. Remove supernatant, leaving the beads covered by liquid.

  • 16.

    Wash the IP beads three times with 5 mL of wash buffer 2. Pellet beads by centrifugation for 5 min at 120 x g and 4 °C (see Note 5). Remove supernatant, leaving the beads covered by liquid.

  • 17.

    Transfer IP beads from the 15 mL conical tube to a 1.5 mL LoBind tube using a blunted pipette tip. Centrifuge for 5 min at 120 x g and RT. Remove supernatant, leaving the beads covered by liquid, and place LoBind tube on ice.

  • 18.

    Make a fresh solution of elution buffer (see Notes 6 and 7).

  • 19.

    Elute KRAS4B from beads using 250 μL of elution buffer at 900 rpm for 20 min at RT (ThermoMixer). Check at 5-min intervals to ensure that beads are evenly resuspended, mixing with a blunted pipette tip if settled. Transfer eluent to new 1.5 mL LoBind tube and store on ice.

  • 20.

    Repeat the elution step two to three times, keeping each recovered fraction separate in a clean LoBind tube on ice.

  • 21.

    Take separate gel aliquots from all elutions (e.g., E1 and E2). Add 50 μL of gel sample loading buffer to the remaining IP beads. Store at — 20 °C or — 80 °C.

  • 22.

    Prepare a 1.5 mL LoBind tube for the ZipTip elution by adding 5 μL of C4 ZipTip elution buffer and storing on ice.

  • 23.

    Prepare for final sample dilution and vial conditioning by chilling HPLC Solvent A on ice.

  • 24.

    Concentrate and desalt the eluted KRAS4B protein via C4 ZipTip. First, add a C4 ZipTip to a p10 pipette, and activate in six 10 μL volumes of the C4 ZipTip activation buffer, dispensing each to waste. Wash the C4 ZipTip in six 10 μL volumes of the C4 ZipTip wash buffer, dispensing each to waste. Transfer the C4 ZipTip to the end of a p200 tip, and bind KRAS4B by pipetting 200 μL of each elution fraction up and down ten times, taking extreme care to not pass air through the resin. Combine multiple elution fractions onto one C4 ZipTip by repeating this step for each fraction collected (e.g., E1, followed by E2). Return the C4 ZipTip to the p10 pipette, and wash in ten 10 μL volumes of the C4 ZipTip wash buffer, dispensing each to waste. Elute KRAS4B from C4 ZipTip by pipetting 5 μL C4 ZipTip elution buffer up and down at least ten times, taking extreme care to not pass bubbles through the resin. Dilute 1:5 with HPLC Solvent A (see Notes 6 and 8).

  • 25.

    Remove BSA from autosampler vials, and wash four times in 200 μL of ice-cold HPLC Solvent A, dispensing to waste each time.

  • 26.

    Transfer sample from LoBind tube containing the final sample from Step #24 into autosampler vial (see Note 9).

3.2. KRAS4B Magnetic Bead Immunoprecipitation (Fig. 2)

Day 1

  1. Wash 75 μL (150 μL of 50% slurry) of protein A/G magnetic beads in 1 mL of 1 × TBS in a 2 mL tube. Pellet beads using a magnet and remove supernatant. Repeat twice more.

  2. Resuspend beads in 1 mL of 0.1 mg/mL BSA in 1X TBS.

  3. Add 15 μg of α-v-HRAS antibody to the bead solution. Incubate for 4 h at RT with constant rotation (see Note 10).

  4. Following incubation, use a magnet to pellet beads at the bottom of the tube, and remove as much of the supernatant as possible while still leaving a visible layer on top of the beads. Wash beads two times in 1 mL of 1 × TBS, each time pelleting the beads on the bottom of the tube and removing as much of the buffer as possible while still leaving a visible layer on top of the beads. Add 1 mL of 1 × TBS and transfer beads from 2 mL tube to a 15 mL tube. Pellet beads at the bottom of the tube and remove supernatant. Equilibrate beads in 1 mL of lysis buffer for 30 min on ice.

  5. Prepare lysis buffer and chill on ice. Add HALT Inhibitor Cocktail only to chilled buffer immediately prior to lysis.

  6. For cell culture (~1 × 108 cells recommended), rinse cells twice with ice-cold PBS, and store on ice. Add ~1 mL of lysis buffer per ~1 × 107 cells (for a 15 cm cell culture dish, this is approximately 2 mL per dish). For cell pellets, thaw cells on ice, followed by resuspension in 8 mL chilled lysis buffer.

  7. For cryopreserved tissue samples (>200 mg recommended), maintain in liquid nitrogen (LN2) until just before cryogrind- ing. Subject to two rounds of grinding (frequency 30 Hz, 2 min), keeping samples immersed in LN2 between rounds. Transfer pulverized lysate to a 50 mL conical tube pre-chilled in LN2. Allow lysate to thaw on ice, followed by resuspension in 25 mL chilled lysis buffer.

  8. Incubate lysate on ice for 30 min. Pipette the lysate every 10 min to ensure even resuspension. Do not vortex. If lysing on dish, use a cell scraper and serological pipette to transfer lysate to a clean conical tube.

  9. Sonicate the lysate on ice at 50% amplitude for 30 2-s cycles, with incubation on ice for 5 s in between each cycle.

  10. Clarify the lysate by centrifugation for 30 min at 16,000 x g and 4 °C.

  11. Take gel aliquots (for western blot) from the lysate supernatant, and store at —20 °C or —80 °C (see Note 2).

  12. Remove the majority of lysis buffer while still leaving a visible layer of buffer on top of the beads (see Note 1).

  13. Add lysate to the IP beads. For lysate from tissue samples (25 mL), divide evenly between two aliquots of IP beads (12.5 mL/50 μL IP beads). Incubate for 16–20 h at 4 °C with constant rotation.

  14. Prepare one autosampler vial per IP by adding 200 μL of 2 mg/mL BSA. Store vial in a closed container at 4 °C until use during Day 2 of IP (see Note 3).

Day 2

  • 15.

    Prepare IP wash buffer 1 and 2. Chill on ice.

  • 16.

    Collect lysate flow through by pelleting beads by magnet (see Note 4).

  • 17.

    Take gel aliquots from the flow through supernatant and store at —20 °C or —80 °C.

  • 18.

    Remove supernatant, leaving the beads covered by liquid (see Note 1).

  • 19.

    Wash the IP beads three times with 5 mL of wash buffer 1. Pellet beads by magnet. Remove supernatant, leaving the beads covered by liquid.

  • 20.

    Wash the IP beads three times with 5 mL of wash buffer 2. Pellet beads by magnet. Remove supernatant, leaving the beads covered by liquid (see Note 5).

  • 21.

    Transfer IP beads from the 15 mL tube to a 1.5 mL LoBind tube using a blunted pipette tip. Pellet with magnet. Remove supernatant, leaving the beads covered by liquid.

  • 22.

    Make a fresh solution of elution buffer (see Notes 6 and 7).

  • 23.

    Elute KRAS4B from beads using 250 μL of elution buffer at 900 rpm for 20 min at RT (ThermoMixer). Check at 5-min intervals to ensure that beads are evenly resuspended, mixing with a blunted pipette tip if settled. Transfer eluent to new 1.5 mL LoBind tube and store on ice.

  • 24.

    Repeat elution step two to three times, keeping each recovered fraction separate in a clean LoBind tube on ice.

  • 25.

    Take separate gel aliquots from all elutions (e.g., E1 and E2). Add 50 μL of gel sample loading buffer to the remaining IP beads. Store at —20 °C or —80 °C.

  • 26.

    Prepare a 1.5 mL LoBind tube for the ZipTip elution by adding 5 μL of C4 ZipTip elution buffer and storing on ice.

  • 27.

    Prepare for final sample dilution and vial conditioning by chilling HPLC Solvent A on ice.

  • 28.

    Concentrate and desalt the eluted KRAS4B protein via C4 ZipTip. First, add a C4 ZipTip to a p10 pipette, and activate in six 10 μL volumes of the C4 ZipTip activation buffer, dispensing each to waste. Wash the C4 ZipTip in six 10 μL volumes of the C4 ZipTip wash buffer, dispensing each to waste. Transfer the C4 ZipTip to the end of a p200 tip, and bind KRAS4B by pipetting 200 μL of each elution fraction up and down ten times, taking extreme care to not pass air through the resin. Combine multiple elution fractions onto one C4 ZipTip by repeating this step for each fraction collected (e.g., E1, followed by E2). Return the C4 ZipTip to the p10 pipette, and wash in ten 10 μL volumes of the C4 ZipTip wash buffer, dispensing each to waste. Elute KRAS4B from C4 ZipTip by pipetting 5 μL C4 ZipTip elution buffer up and down at least ten times, taking extreme care to not pass bubbles through the resin. Dilute 1:5 with HPLC Solvent A (see Notes 6 and 8).

  • 29.

    Remove BSA from autosampler vials, and wash four times in 200 μL of ice-cold HPLC Solvent A, dispensing to waste each time.

  • 30.

    Transfer sample from LoBind tube containing the final sample from Step #28 into autosampler vial (see Note 9).

3.3. LC-MS/MS Parameters for Targeted Top-Down Characterization of KRAS4B

  1. KRAS4B proteoforms should be further resolved by reverse- phase nanocapillary liquid chromatography (LC) prior to introduction into the mass spectrometer. The parameters provided herein are for a Dionex Ultimate 3000 LC system coupled with a Q-Exactive HF BioPharma mass spectrometer (Thermo Fisher Scientific).

  2. The LC should use the described (Methods) trap and nanocapillary analytical columns coupled to a vented tee setup and nanospray emitter [10]. For the alternative commercial columns and source listed in Methods, employ a trap-in-valve configuration and an extender line to the NanoFlex ion source, with parameters as described in [11].

  3. Maintain the LC temperature at 45 °C throughout LC-MS/ MS analysis. Load an injection volume of 5 μL of KRAS4B sample (prepared by IP less than 24 h prior to LC-MS/MS, with samples prepared immediately prior to LC-MS/MS recommended; see Note 9) onto the trap column, and wash in HPLC Solvent A for 10 min at a flow rate of 2.5 μL/min. Elute KRAS4B proteins into the mass spectrometer at a flow rate of 0.3 μL/min by the following gradient: 5% HPLC Solvent B at 0 min, 30% Solvent B at 5 min, 45% Solvent B at 25 min, 95% Solvent B from 28–31 min, and 5% Solvent B from 34 to 50 min.

  4. Acquire partial intact mass (MS1) spectra using a 200 m/z window to determine both the most abundant charge states of KRAS4B and the location of KRAS4B within the chromatogram. This step can be performed simultaneously with step 5 within a single LC-MS/MS run.

  5. MS1 scan method parameters:
    1. Protein Mode.
    2. Resolving power (r.p.): 120,000 (at 200 m/z).
    3. Scan window: 750–950 m/z.
    4. Average of four microscans.
    5. Automatic gain control (AGC) target: 1E+06.
    6. Maximum ion injection time: 50 ms.

  6. Acquire targeted intact mass (tMS1) spectra using a selected ion monitoring (SIM) method targeting individual charge states of KRAS4B (e.g., 23+, Table 1). SIM focuses the maximum of allowed ion current through a narrow (10 m/z) window, providing a dramatic increase in sensitivity and facilitating detection of low-abundance endogenous KRAS4B proteoforms.

  7. tMS1 scan method parameters:
    1. Protein Mode.
    2. Resolving power (r.p.): 120,000 (at 200 m/z).
    3. Isolation window: 10 m/z (e.g., 920.0–930.0 for the KRAS4B 23+ charge state).
    4. Average of four microscans.
    5. Automatic gain control (AGC) target: 5E+04.
    6. Maximum ion injection time: 400 ms.

  8. Fragment ion (MS2) spectra are used to confirm proteoform sequence and localize post-translational modifications (PTMs). MS2 data is similarly collected by targeting a list of pre-selected values with increasingly narrow m/z windows to provide diagnostic fragment ions to be used in proteoform quantitation and comparison (tMS2).

  9. For tMS2 target peak selection, open the Global Parameters tab in the method editor. Select Inclusion List from the dropdown menu, and then enter the m/z value, charge, and retention time range for each proteoform target. It is recommended that three or fewer abundant proteoforms be targeted per retention time range using the given parameters.

  10. tMS2 scan method parameters:
    1. Protein Mode.
    2. Default Charge: 23+ (value corresponding to example targeted charge state).
    3. r.p.: 60,000 (at 200 m/z),
    4. Isolation window: 8 m/z (wide) or 3 m/z (narrow).
    5. Average of four microscans.
    6. Minimum m/z: 400.
    7. AGC target: 1E+06.
    8. Maximum ion injection time: 800 ms.
    9. High-energy collisional dissociation (HCD) normalized collision energy (NCE): applied in 2% steps between 19 and 25%.
  11. Additional MS parameters:
    1. Heated transfer capillary temperature: 320 ”C.
    2. S-lens RF amplitude: 50%.
    3. 15 V in-source dissociation.

Table 1.

KRAS4B charge states and their expected tMS1 (10 m/z) windows

KRAS4B charge state tMS1 (10 m/z) window
23 920–930
24 881–891
25 846–856
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3.4. Data Analysis (see Note 11; Figs. 3 and 4)

Fig. 3.

Fig. 3

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Example intact mass (MS1) spectra (top) and MS2 graphical fragment maps (bottom) supporting the precise characterization of intact KRAS4B proteoforms: PFR 249914, PFR 249915, PFR 249917, and PFR249918 in HCT-116 Par (WT/G13D) cells by the SOP IP protocol

Fig. 4.

Fig. 4

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Visual guide on how to use ProSight Lite [9] to characterize KRAS4B proteoforms from MS1 and MS2 data collected during TDMS

  1. To analyze the data in ProSight Lite, the monoisotopic masses for both the precursor and the fragmentation scans will be needed.

  2. Open the .raw file in Xcalibur QualBrowser.

  3. Average across the most abundant portion of the MS1 chromatographic peak corresponding to the KRAS4B proteoform of interest. Click Export and then the Xtract function. Select the following parameters for deconvolution and deisotoping:
    1. Generate mass mode: M.
    2. Resolution@400: 120000.0.
    3. S/N threshold: 3.
    4. Max charge: 30.
    5. Averagine table: Averagine.

  4. A new window will appear. Use the left and right keyboard arrow keys to reach Scan #2 of PostXtract results, where the monoisotopic, zero-charge (13C0) mass spectrum can be found. Click Export and then Clipboard (Exact Mass). Paste into a spreadsheet (i.e., Microsoft Excel), and save the file with a title including MS1.

  5. Repeat steps 1-3 but with the HCD fragmentation data files and the Resolution@400 set to 60,000. Save the spreadsheet with a title including MS2.

  6. Open ProSight Lite (Fig. 4) [9].

  7. Select Add Experimental Data on the home page of ProSight Lite. Copy the monoisotopic mass from the MS1 spreadsheet, and paste under Precursor. Copy Mass data from the MS2 spreadsheet file and paste under Fragments. Set the following parameters:
    1. Precursor mass type: Monoisotopic.
    2. Mass mode: M (neutral).
    3. Fragmentation methods: HCD.
    4. Fragmentation tolerance: 10 ppm.

  8. Copy and paste the KRAS4B sequence under Add Candidate Sequence tab on the home page of ProSight Lite (can be found at https://www.uniprot.org/uniprot/P01116–2.fasta). From the sequence, you will need to remove the N-terminal methionine (M) and the C-terminal valine (V), isoleucine (I), and methionine (M) residues. For Fixed Modifications, use No Modification for cysteine and methionine.

  9. Common modifications include adding an acetylation modification to the N-terminus (Thr 2 of KRAS4B) as well as a farnesylation and/or carboxymethylation to the C-terminus (Cys 185 of KRAS4B, proteoform-dependent). Common and custom modifications can be added to residues within the sequence as required.

  10. Detailed explanation of the results can be found in DeHart et al. [12]. In short, the Mass Diff. between the Theoretical Mass (calculated from sequence and modification masses) and the Observed Mass (from MS1 spreadsheet file) is reported in both Da and ppm. The p-score describes the probability of the fragments matching the proteoform due to random chance; the smaller the p-score, the less likely it is that the fragments match the proteoform due to random chance [13].

4. Notes

  1. Curtail oxidation of KRAS4B proteoforms by limiting exposure of IP beads to the air. It is recommended that a thin layer of buffer be maintained on top of the IP beads during all steps of the IP protocols.

  2. It is possible that the IP may fail. A good way to assess the points of failure is to perform a western blot of the IP fractions using an a-RAS primary antibody (Fig. 5). All bands corresponding to KRAS4B should be around the 21 kDa molecular weight marker.
    1. If lysis was unsuccessful, a faint band or absence of one will occur in the input (IN) fraction. To fix this, try an increased volume of lysis buffer and/or additional repetitions of sonication. It is also possible that the starting concentration of cells or tissue used was not sufficient to detect KRAS4B proteoforms via IP-TDMS. If this is the case, increase the concentration of starting material.
    2. The flow through (FT) fraction should have a minimal band or no band present. If a strong band is present, increase the ratio of IP beads to lysate volume.
    3. The elution fraction (E) should have a faint to strong band. If no band is present, the elution was unsuccessful. The pH of the elution may not have been acidic enough if too thick of a layer of wash buffer 2 was left on top of the IP beads when the elution buffer was added. This can be avoided by removing as much liquid as possible from the covered beads immediately prior to elution and ensuring that the elution buffer is close to pH 2 (e.g., by dispensing an aliquot onto pH paper) prior to the elution step.
    4. To verify complete elution of KRAS4B from the IP beads, a boiled beads control can be incorporated into the diagnostic immunoblot. This control may have KRAS4B remaining if the elution steps did not work properly, if more elution steps are needed, or if the elution composition needs to be altered depending on the KRAS4B pro- teoform population. For populations with more hydrophobic KRAS4B, consider eluting with a buffer composed of 0.5% TFA and 10% ACN.

  3. Coat all LC-MS/MS vials in 2 mg/mL BSA for at least 4 h prior to use. This reduces loss ofhydrophobic KRAS4B proteo- forms on the sides of the LC vial.

  4. Limit loss of IP beads throughout the protocols by paying special attention to the progress of bead pelleting. If the supernatant has a considerable quantity of agarose IP beads still present following centrifugation, repeat the centrifugation step, or leave the tube on ice until fewer beads remain in the supernatant. For magnetic IP beads, the supernatant will have a brown tint if too many IP beads remain. Continue pelleting magnetic IP beads with a magnet until the supernatant is clear.

  5. If the concentration of polymer contaminants from residual detergent in the final sample precludes KRAS4B proteoform characterization, perform three to five additional wash steps with IP wash buffer 2.

  6. Use only Optima-grade ACN and H2O for all buffers during and after the elution steps. Do not store these reagents in plastic.

  7. Use only fresh TFA when preparing the elution buffers. It is recommended to use single-use 1 mL ampules of concentrated TFA when preparing buffers and to make the elution buffer immediately prior to the elution step. Do not store concentrated TFA in plastic tubes.

  8. To increase the quantity of KRAS4B proteoforms in the final elution, consider performing the C4 ZipTip protocol twice. Elute KRAS4B from both C4 ZipTips into the same final 5 μL elution.

  9. The KRAS4B proteoforms can last at 4 °C for 24 h post-final elution; however, considerable loss in signal will occur after 24 h. Do not freeze and thaw samples, or delay sample preparation at any step throughout the IP protocols. It is highly recommended that LC-MS/MS analysis be performed imme-diately following completion of the C4 ZipTip cleanup and final dilution steps. If this is not feasible, samples should be stored in washed, BSA-conditioned vials on ice at 4 °C until they can be run.

  10. Not all antibodies are created equal. Keep this in mind when using antibodies other than the anti-v-HRAS antibody used in this protocol. Antibody concentration, magnetic bead epitope (i.e., Protein A/G, Protein G, etc.), incubation times, and temperatures may need to be optimized accordingly.

  11. For a more exhaustive explanation of how to analyze top-down proteomics data using Xtract and ProSight Lite, refer to DeHart et al. [12].

Fig. 5.

Fig. 5

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Visualization of an example SOP IP by SDS-PAGE and subsequent a-Pan RAS (abcam 52939) immunoblot. IP fractions shown include the input/clarified whole cell lysate (IN; 1:1000), flowthrough (FT; 1:1000), elution (E1; 1:100), and molecular weight marker (MW). The far left lane contains 50 ng of recombinant processed KRAS4B standard (Std) to mark the expected MW and estimate the quantity of recovered RAS within the IP elution. Note that a band is still present in the FT fraction, suggesting that not all KRAS4B was depleted from the input. Increasing the IP bead to cell lysate protein concentration ratio can improve depletion

Acknowledgments

We thank Kevin Haigis for providing an agarose bead-based IP protocol and the following coauthors of the Ntai et al. [7] manuscript for their significant contributions to the initial IP-TDMS method development: Ioanna Ntai and Luca Fornelli. This work was supported by federal funds from the National Cancer Institute (Office of Cancer Clinical Proteomics Research), National Institutes of Health, under Contract HHSN261200800001E, and Lei- dos Biomedical Research under Contract HHSN261200800001E and was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under National Institutes of Health Grant P41 GM108569. L.M.A. is supported by T32GM008382.

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