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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Methods Mol Biol. 2022;2500:15–30. doi: 10.1007/978-1-0716-2325-1_3

Size Exclusion Chromatography Strategies and MASH Explorer for Targeted Proteoform Characterization

Timothy N Tiambeng 1, Zhijie Wu 1, Jake A Melby 1, Ying Ge 1,2,3,*
PMCID: PMC9703982  NIHMSID: NIHMS1841155  PMID: 35657584

Abstract

Top-down mass spectrometry (MS)-based analysis of larger proteoforms (> 50 kDa) is typically challenging due to an exponential decay in the signal-to-noise ratio with increasing protein molecular weight (MW) and co-elution with low-MW proteoforms. Size exclusion chromatography (SEC) fractionates proteins based on their size, separating larger proteoforms from those of smaller size in the proteome. In this protocol, we initially describe the use of SEC to fractionate high MW proteoforms from low MW proteoforms. Subsequently, the SEC fractions containing the proteoforms of interest are subjected to reverse phase liquid chromatography (RPLC) coupled online with high-resolution MS. Finally, proteoforms are characterized using MASH Explorer, a user-friendly software environment for in-depth proteoform characterization.

Keywords: Size exclusion chromatography, proteoforms, data analysis, Top-down proteomics

Introduction:

Top-down mass spectrometry (MS)-based proteomics is a powerful technology due to its capability to characterize proteoforms arising from sequence variations, alternative splicing, and post-translational modifications (PTMs) [1–8]. Despite the capability of top-down proteomics, high molecular weight (MW) proteoforms are often under-represented in the MS analysis of the proteome [9–11]. This challenge arises from both the high dynamic range of the proteome, in which protein expression can vary in orders of magnitude, and the exponential decay in the MS signal-to-noise ratios of large proteoforms, resulting mainly from the increased number of charge states observed with electrospray ionization of proteoforms, the greater contribution of heavy isotopes at higher precursor mass, and the detrimental influence of adducting and interfering species [12]. MS-identification and characterization of large proteoforms are particularly challenging if they are co-eluted with smaller proteoforms [9,10]. Moreover, user-friendly software tools remain under-developed for proteoform identification and comprehensive characterization of large proteoforms.

Size exclusion chromatography (SEC) addresses a critical challenge in top-down MS-based analysis of large, high-MW proteoforms[9,10,13,14]. This chromatographic technique fractionates the proteome by separating proteoforms based on their size or hydrodynamic volumes, while reducing sample loss due to minimal interactions of proteins with the SEC stationary phase [13,14]. SEC is usually performed using hydrophilic stationary phases with well-defined pore diameters [15]. Typically, smaller proteoforms more readily diffuse into the pores, while relatively larger proteoforms are less likely to enter the pores. Thus, proteoforms separate based on their size as they pass through the column and are eluted in order of decreasing molecular weight [16]. SEC can be used as an effective and versatile separation method that is highly compatible with top-down MS-based proteomics and is orthogonal to other chromatographic methods. SEC experiments can be performed in both denatured and native modes, using mobile phases such as formic acid in water [10] and ammonium acetate solution [17], respectively. The resulting SEC fractions containing proteoforms eluting at specific chromatographic elution times can then be subjected to high-resolution MS with simple sample processing. As a notable example, Cai and Tucholski et. al., developed serial size exclusion chromatography (sSEC) by connecting together SEC columns in series with different stationary phase pore diameters, to achieve efficient size-based separation over a broad MW range [9]. When sSEC fractions were further separated by RPLC coupled online with high resolution MS, it enabled a 15-fold improvement in the observation of proteoforms greater than 60 kDa compared to one-dimensional RPLC-MS alone. Additionally, Tucholski et. al., utilized sSEC fractionation directly with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) analysis to enable sequence characterization of large proteoforms without RPLC separation or extensive protein purification [10].

For characterizing the primary sequence and localizing the PTMs of proteoforms, MASH Explorer is a universal, user-friendly, and freely available software environment for top-down proteomics [18]. MASH Explorer is built upon the previous successes of MASH Suite [19] and MASH Suite Pro [20], to bolster the continued growth of the top-down proteomics community. In comparison to MASH Suite Pro, MASH Explorer can process MS, tandem MS (MS/MS), and liquid chromatography tandem MS (LC-MS/MS) across multiple vendor-specific-formats, with automated database searching for protein identification, and tools for proteoform characterization and data validation. MASH Explorer incorporates various deconvolution and database searching algorithms to assist fragment ion identification and large proteoform characterization. MASH Explorer provides an effective and comprehensive solution to process many types of MS data, and can be used in combination with other freely available software tools, such as Proteoform Suite [21,22], to assist identification of large proteoforms [11] that are not monoisotopically resolved.

Here in this protocol, we demonstrate that SEC can be generally used to fractionate high-MW proteoforms from low-MW proteoforms. SEC fractions are then subjected to online LC-MS and LC-MS/MS analysis using a quadrupole time-of-flight (QTOF) instrument. Subsequently, MASH Explorer [18] is used to process and analyze MS data to characterize the sequence and PTMs of proteoforms.

2. Materials

All reagents were purchased from MilliporeSigma Inc. (St. Louis, MO, USA) unless otherwise noted. HPLC grade solvents, such as water, acetonitrile, ethanol, isopropanol, and formic acid were purchased from Fisher Scientific (Fair Lawn, NJ, USA) (see Note 1).

2.1. Size Exclusion Chromatography

  1. Waters ACQUITY H-Class UPLC system equipped with a UV detector and an automatic fraction collector (Waters Corporation, Milford, MA, USA).

  2. PolyHYDROXYETHYL A (PolyHEA) columns (PolyLC Inc., Columbia, MD, USA) (see Note 2).

  3. Mobile phase, such as 1% formic acid in water (v/v) (see Note 3).

  4. Ultra-centrifugal 10 kDa molecular weight cutoff (MWCO) filters (0.5 mL) (Fisher Scientific, Fair Lawn, NJ, USA).

  5. 1/16” O.D. (outer diameter) PEEK tubing with 100 μm I.D. (inner diameter), 2 cm (VICI Valco instruments, Houston, TX, USA)

  6. Supelco® 1/16” O.D. PEEK ferrules and fittings (MilliporeSigma Inc. (St. Louis, MO, USA)

2.2. Online Reverse Phase Liquid Chromatography Mass Spectrometry

  1. maXis II quadrupole time-of-fight (QTOF) mass spectrometer (Bruker Daltonics, Bremen, Germany).

  2. Waters nanoACQUITY UPLC system (Waters Corporation, Milford, MA, USA).

  3. Home-packed PLRP column, (200 × 0.5 mm I.D with PLRP-S bulk media (10 μm, 1000 Å, Agilent Technologies, Santa Clara, CA, USA) (See Note 4)

  4. 1/16” O.D. PEEK tubing with 100 μm I.D., 20 cm (VICI Valco instruments, Houston, TX, USA)

  5. Supelco® 1/16” O.D. PEEK ferrules and fittings (MilliporeSigma Inc. (St. Louis, MO, USA)

  6. Mobile Phase A: 0.1% formic acid in water (v/v).

  7. Mobile Phase B: 0.1% formic acid in 50:50 acetonitrile/ethanol (v/v).

  8. Absorbance microplate reader, such as the BioTek ELx808 Absorbance Reader (Winooski, VT, USA).

  9. Bradford protein assay reagents, such as the Quick Start™ Bradford Protein Assay Kit (Hercules, CA, USA).

2.3. MASH Explorer Data Analysis

  1. Personal computer meeting the minimum hardware requirements (See Note 5).

  2. MASH Explorer software, version 2.1.1. (https://labs.wisc.edu/gelab/software.html).

  3. MASH Explorer User Installation Guide: (https://labs.wisc.edu/gelab/MASH_Explorer/doc/User%20Installation%20Guide.pdf) (See Note 6)

3. Methods

3.1. Size Exclusion Chromatography

  1. For new and previously unused PolyHEA columns, flush the column with 15 column volumes of water to remove methanol.

  2. Condition the column(s) by flushing with the intended mobile phase overnight at room temperature to ensure reproducible retention times.

  3. For serial SEC, connect the desired number of SEC columns together using 2 cm segments of 1/16” O.D. PEEK tubing (100 μm I.D.), and 1/16” O.D. PEEK ferrules and fittings.

  4. Ensure that an appropriately sized sample loop is installed on the UPLC system for SEC or serial SEC applications (See Note 7)

  5. Set the flow rate to 0.5 mL/min and perform three sample injections using water at the same flow rate and isocratic gradient as the intended sample to ensure stable baseline and column backpressure. Ensure that the sample manager is kept at 4°C to reduce artifactual temperature-induced protein modification, such as oxidation. (See Note 8)

  6. As standards for reproducibility, inject 5 μg of protein solution that is buffer exchanged with the starting SEC mobile phase. (See Note 9)

  7. Monitor protein elution from the SEC column by recording the change in UV-absorbance at 280 nm as a function of retention time. (See Note 10)

  8. For fraction collection, begin collecting fractions when the absorbance intensity increases about 10 times the signal-to-noise ratio, or at the approximate elution time of the protein of interest based on the elution times of the standard proteins (See Note 11). Ensure that the fraction manager is kept at 4 °C to reduce artifactual temperature-induced protein modification.

  9. Concentrate the desired protein-containing SEC fractions using 10 kDa MWCO (See Note 12) for SDS-PAGE (See Note 13) and LC-MS/MS analysis (Fig. 1).

Fig. 1.

Fig. 1

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Example of an SEC separation of a complex protein mixture. Comparison of (A) single column SEC (500 Å), (B) SEC with two columns connected serially (1000 Å–500 Å), and (C) SEC with three columns connected serially (1000 Å–500 Å–500 Å) for the fractionation of the same protein loading mixture (LM). The top panel illustrates the UV chromatogram for the corresponding SEC experiment, annotated with numbers corresponding to the collected SEC fractions. The bottom panel illustrates the SDS-PAGE analysis corresponding to the collected and annotated SEC fractions (Adapted and reprinted with permission from [9] Copyright 2017 American Chemical Society.)

3.2. Online Reverse Phase Chromatography and Top-down MS Analysis:

  1. Prepare LC-MS grade 0.1% formic acid in water (v/v) and LC-MS grade 0.1% formic acid in 50:50 acetonitrile:ethanol (v/v).

  2. Dilute the protein fractions to a total protein concentration of 100 ng/μL total protein with 0.1% formic acid in water containing 2 mM TCEP. (See Note 14)

  3. Load 500 ng of total protein for a 0.5 mm I.D. PLRP-S column. Separate the proteins chromatographically using a nanoAcquity UPLC system equipped with a PLRP-S column. Connect the PLRP-S column to the electrospray ionization source using 1/16” O.D. PEEK tubing with 100 μm I.D., and 1/16” O.D. PEEK ferrules and fittings.

  4. Elute proteins using a mobile phase gradient going from 5% B to 95% B over 45 min at a flow rate of 8 μL/min (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in 50:50 acetonitrile:ethanol). (See Note 15)

  5. For the electrospray ionization source on the QTOF mass spectrometer, set the “End Plate Offset” at 500 V, the “Capillary” at 4500 V, the “Nebulizer” at 0.5 bar, the “Dry Gas” at 4.0 L/min, and the “Dry Temp” at 220 °C. Collect mass spectra at a scan rate of 0.5 Hz over 500–2000 m/z range.

  6. Complete an initial LC-MS run on a SEC protein fraction of interest and perform data analysis to determine the charge state ions, molecular weight, and retention time of each target protein.

  7. For targeted protein analysis, complete a second LC-MS run using online collisionally activated dissociation (CAD) by inputting the precursor ion(s), ion isolation range (m/z), CAD energy, and the chromatographic elution interval. The precursor ion(s) will be isolated and fragmented in the targeted MS/MS experiment for protein identification (Fig. 2) (See Note 16).

Fig. 2.

Fig. 2

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Top-down targeted MS/MS for protein identification in a selected sSEC fraction. (A) CAD-based MS/MS experiments are performed on selected LC retention time windows containing proteoforms of interest. (B) High-resolution MS demonstrating representative precursor ion isolation and effective CAD-based fragmentation of a 42.9 kDa protein, yielding high-resolution tandem mass spectra. (Adapted and reprinted with permission from [9] Copyright 2017 American Chemical Society.)

3.3. Data Analysis using MASH Explorer’s Targeted Mode:

  1. Import raw MS/MS data files into the MASH Explorer software under Targeted Mode (Fig. 3), (See Note 17).

  2. Perform spectral deconvolution using algorithms such as THRASH [23] or TopFD [24] (See Note 18). After selecting a deconvolution method, proceed to the Advanced tab to change settings such as the precursor ion m/z and charge in the General subheading, as well as specific algorithm parameters such as Max Charge and Max Mass in the Deconvolution subheading. A list of deconvoluted fragment ions will be generated by the selected algorithm and automatically imported back to the MASH Explorer software interface. (Fig. 4)

  3. Verify the deconvoluted fragment ions in the Mass List by adjusting their charge states and monoisotopic mass. A more precise list of fragment ions will allow for shorter processing time and more accurate proteoform identifications in the database search.

  4. Using the verified fragment ion list, perform database search using algorithms such as MS-Align+ [25] (See Note 19). The precursor ion information obtained during data analysis in section 3.2, such as the precursor ion m/z and charge will assist the database search algorithm to identify targeted proteoforms more accurately.

  5. Import the identified proteoform identification after completing the database search. Under the Characterization heading, select Import Database Search Results, and select the database algorithm used for database search (e.g., TopPIC Search Results). Select the folder labeled “MASH_Workflows” and select the appropriate MassList folder labeled with the database search algorithm (e.g., ‘…MassList_TopPIC_1_1) (Fig. 5). The folder contains a .CSV file with the Protein Search results. After opening the .CSV file, the protein sequence will be uploaded to MASH Explorer’s Sequence Table.

  6. Using MASH Explorer software, visualize the verified fragment ion list against the imported proteoform sequence in the Sequence Table for proteoform characterization (Fig. 6). A warning message will appear from MASH Explorer reminding the user to correct the protein sequence to obtain the correct fragmentation list.

  7. Characterize the protein post-translational modifications. Users can obtain this information by intact mass analysis during section 3.2, or by database search algorithm results (See Note 20).

  8. Use database search algorithms in MASH Explorer to identify proteoforms (See Note 21).

Fig. 3.

Fig. 3

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MASH Explorer’s Targeted Mode interface for MS/MS data import.

Fig. 4.

Fig. 4

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MS/MS spectral deconvolution and the calculated mass list imported by MASH Explorer software from the selected deconvolution algorithm, displayed in MASH Explorer’s Mass List panel (highlighted in red).

Fig. 5.

Fig. 5

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MASH Explorer enables import of database search results. A) MASH Explorer supports multiple database search algorithms including TopPIC [24], MS-Align+ [25], pTop [26], and Informed-Proteomics [27] workflows. B) Highlighted .CSV file containing database search results from the “MASH_Workflows” folder.

Fig. 6.

Fig. 6

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Visualizing and verifying fragment ions against an imported protein sequence in MASH Explorer. A) Imported database search results can be found under the Protein Search Result tab. B) MASH Explorer’s ‘Protein Sequence Table’ allows users to visualize fragment ion mapping for different MS/MS techniques to characterize the protein sequence and PTMs. The protein amino acid sequence corresponding to the identified protein is directly imported from the database search. Correcting the protein sequence according to the database search (N-terminal methionine excision), results in significantly improved ion fragmentation lists. C) MASH Explorer enables manual verification of MS/MS fragment ion data and adjustment of charge state and monoisotopic mass.

Acknowledgements:

We would like to thank Trisha Tucholski for helpful discussions. This work was supported by NIH R01 GM117058 and NIH R01 GM125085 (to Y.G.). Y.G. also would like to acknowledge R01 HL096971 and S10 OD018475. T.N.T. would like to acknowledge support from the NIH Chemistry-Biology Interface Training Program NIH T32GM008505. J.A.M. would like to acknowledge support from the Training Program in Translational Cardiovascular Science, T32 HL007936-20.

4. Notes:

1)

Ensure that all mobile phases are prepared in thoroughly cleaned glassware intended for HPLC solvents. Always clean glassware with the same mobile phase/solvent used for the chromatography; never wash glassware with detergent as this can introduce contaminants during LC-MS analysis.

2)

A variety of column dimensions can be used for SEC and sSEC experiments. Typical column dimensions include 200 mm x 9.4 mm, 3 μm particles with pore sizes of 500 Å, and 1000 Å. Ideally for SEC, the use of small particles, wider I.D. columns, and shorter column lengths can result in columns where wall effects are negligible and protein band spreading decreases [28]. Strategies such as minimizing sample injection volume and increasing initial protein loading can assist in reducing significant dilution and loss of UV signal during SEC.

3)

A variety of MS-compatible mobile phase compositions can be used for the fractionation of different protein-classes. Previously, we have demonstrated that 1% formic acid is effective for acid-soluble and water-soluble proteins [9,10]. For membrane protein fractionation, our lab has shown that addition of 40% isopropanol (1% formic acid, 40% isopropanol, 59% water v/v) can facilitate membrane protein solubility in the absence of surfactants [29]. For proteins that strongly interact with each other, even under denaturing conditions, the addition of hexafluoroisopropanol can be helpful to disrupt protein-protein interactions and can be added to a final concentration of 100 mM. Samples with hexafluoroisopropanol can be centrifuged in MWCO filters at 15,000 x g for 30 min at 4 °C prior to SEC.

4)

The maximum backpressure of this PLRP-S material is approximately 3000 psi; exceeding this pressure will reduce the performance and lifetime of the column.

5)

MASH Explorer requires Microsoft Windows operating system with installation with .NET framework. It is recommended the personal computer to have at least central processing units with two cores and two threads, and four gigabytes of random-access memory for operation. For deconvolution and database search algorithms, it is suggested that a personal computer with a minimal computing power of a central processing unit with four cores and four threads, and eight gigabytes of random-access memory is used.

6)

The MASH Explorer installation package does not contain any deconvolution or database search algorithms except for the THRASH deconvolution algorithm. To access other supported deconvolution and database search algorithms, they must be downloaded from the individual research groups which developed them. MASH Explorer allows for convenient import of these deconvolution and database search algorithms by accessing the Tools menu and selecting the Configuration dialog.

7)

For single column SEC (200 × 4.6 mm, 5 μm, 500 Å PolyHEA), the protein loading capacity is approximately 500 μg. A sample loop volume of 50 – 250 μl can be used for sample injection. Using smaller sample loop volumes when possible helps to reduce protein band broadening. For single column SEC, we typically use a 50 μl sample loop and 100 μg protein loading at 2 μg/μl sample concentration; for two SEC columns connected serially, we use a 250 μl sample loop and 200 μg protein loading; for three SEC columns connected serially, we use a 250 μl sample loop and 300 μg protein loading. Serial SEC can improve protein separation and fractionation range but requires a larger sample injection and greater protein loading to avoid diluting protein signal.

8)

Using a single SEC column (200 × 9.6 mm, 3 μm, 500 Å PolyHEA), the backpressure is approximately 1000 psi using a flow rate of 0.5 mL/min with 1% formic acid in water (v/v) as mobile phase. For two SEC columns connected serially, the pressure is approximately 1500 psi; for three SEC columns connected serially, the pressure is approximately 2000 psi. Use a KimWipe™ to detect solvent leaks at the junction of the fittings and the columns.

9)

A panel of standard proteins are often used in our lab to evaluate the performance of SEC columns. Depending on native or denaturing SEC applications, these standard proteins include thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), myoglobin (17 kDa). For denaturing SEC, β-amylase, bovine serum albumin, and myoglobin are typical standards used to evaluate the performance of SEC columns.

10)

Proteins usually show UV-absorption maxima between 275 and 280 nm, which are caused by absorbance of the two aromatic amino acids tryptophan (Trp) and tyrosine (Tyr), and to a small extent, by the absorbance of cystine (i.e., disulfide bonds) [30].

11)

We normally collect SEC fractions using 1-min intervals (~ 0.5 mL elution fractions), but longer time intervals (e.g., 1.5-, or 2-min intervals) can be collected and pooled together.

12)

MWCO filters are pre-rinsed by centrifuging with the mobile phase used in the SEC separation to reduce protein loss resulting from concentration of the SEC fractions. 2 mM TCEP is added to reduce protein oxidation and is active under acidic conditions such as 1% formic acid in water (v/v).

13)

For typical SDS-PAGE experiments, commercially available products such as 8–16% Mini-PROTEAN® 12.5% Tris-Glycine eXtended (TGX) gels or Novex™ 8 to 16 % Tris-Glycine Plus Midi Gels can be used according to the manufacturer’s instructions (e.g., 125 V until electrophoresis is complete). For protein loading between 100 – 1000 ng per lane, it is recommended to use a highly sensitive staining method such as SYPRO Ruby or Silver Staining. For protein loading greater than 1 μg per lane we normally perform Coomassie Brilliant Blue staining.

14)

To quantify and normalize total protein loading for equal protein loading in SDS-PAGE and LC-MS experiments, we typically use the Bradford microplate assay for protein samples that are prepared without detergents and measure the absorbance of standards and samples at 595 nm using an absorbance microplate reader. For samples which contain detergents, it is recommended to use the bicinchoninic acid (BCA) microplate assay, which is compatible with most ionic and non-ionic detergents, and measure the absorbance of standards and samples at 562 nm using an absorbance microplate reader.

15)

While LC mobile phase gradients will require optimization and vary depending on the protein mixture, a typical LC gradient used for the separation of cardiac proteins commonly performed in our lab is 0–5 min 20% B, 5–17 min 20–40% B, 17–25 min 40–50% B, 25–32 min 50–65%, 32–43 min 65–95 % B, 43–48 min 95% B, 48–52 min 95–5% B, 52–60 min 5% B.

16)

To select the appropriate fragmentation energy for targeted MS/MS experiments using collisionally activated dissociation (CAD), the precursor ion in the resulting MS/MS spectrum should still be present at 50–70% base peak intensity to avoid over fragmentation, which may result in internal fragments and lower identified fragments. For larger MW proteins, they may require lower fragmentation energy than that required for smaller MW proteins. A good starting range for CAD energy is 10–20 eV for large MW proteins.

17)

To support data import, MASH Explorer uses both ProteoWizard [31] and vendor-specific software (Thermo .RAW, Bruker .BAF, Bruker .ascii, Waters .RAW, .MzXML, .MGF, .mzML) .

18)

MASH Explorer supports several deconvolution algorithms including THRASH, TopFD [24], and pParseTD [26], MS-Deconv [32],. The results from THRASH, MS-Deconv, and TopFD are most often used for fragment ion verification.

19)

MASH Explorer supports multiple database search algorithms including TopPIC [24], MS-Align+ [25], pTop [26], and Informed-Proteomics [27] workflows.

20)

PTMs are supported directly in MASH Explorer software. Modifications such as acetylation, trimethylation, phosphorylation, etc., can be directly added on the target amino acid in the protein sequence. Other modifications can be added using custom modification. Database search algorithms are useful tools in identifying PTMs such as N-terminal acetylation and methylation. However, for phosphorylation, manual proteoform characterization is often needed using the verified fragment ion list [33–40].

21)

Identification of larger MW proteoforms may not be confidently and accurately identified by database search algorithms. Using the verified fragment ion list, a series of fragment ions in a protein sequence may be used to derive a three or four consecutive amino acid sequence, called sequence tag. This sequence tag can be as an alternative method to identify target proteoform matching the intact protein mass. The amino acid sequence for the sequence tag can be derived by mass differences of several fragment ions. The mass differences can be matched to one or the sum of multiple amino acid residue masses (https://proteomicsresource.washington.edu/mascot/help/aa_help.html). The obtained sequence tag can be searched against the database using the guide provided by UniProt (https://web.expasy.org/tagident/).

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