2D Gel Spot Picking and Protein Identification by Mass Spectrometry
A 2D gel tells you which spots changed. It does not tell you what they are. Identification means cutting the spot out of the gel, digesting the protein into peptides and reading those peptides on a mass spectrometer. This guide covers each step: choosing the spots, excising them by hand or with a spot-picking system, in-gel digestion with a sourced protocol, MALDI-TOF and LC-MS/MS identification, and database searching. It ends with how analysis software turns a list of significant spots into a pick list a robot can run.
Choosing which spots to pick
Pick spots on evidence, not on eye. The spots worth identifying are the ones that differ significantly between conditions after proper statistics, and preferably the ones whose q-values, fold changes and expression profiles all point the same way. A well-run analysis ends with a ranked list; that list is the pick list.
Two practical points. First, pick from a preparative gel loaded with more protein than an analytical gel if the spots of interest are faint, because identification needs enough peptide to detect. Second, pick a few abundant, unchanging spots as controls; they confirm that the digestion and MS workflow is working before you trust a negative result on a spot that matters.
Manual spot excision
For a handful of spots, hand cutting works. Print the gel image at actual size with the target spots marked, place it under the gel on a light box, and cut each spot with a clean pipette tip, a cut-down tip, or a purpose-made manual spot cutter. Cut inside the spot boundary so that you take the spot and not its neighbor, and place each plug in a labeled tube immediately.
The manual method’s limits are throughput and traceability. Picking fifty spots by hand is slow and error-prone, and the record of what came from where is a handwritten sheet. Analysis software can help even here: SameSpots exports a picking template with coordinates and a numbered image for exactly this purpose.
Spot picking systems
A spot picker, or spot cutter, is a robot that takes the coordinates of target spots from the analysis software, locates them on the physical gel using reference markers, punches out each plug with a pick head, and deposits it in a numbered well of a microplate. The pick head is typically 1.5 mm or 3 mm in diameter. The gain is not only speed: the plate position is recorded against the spot number, so the chain from gel image to MS result is unbroken.
Pickers have been made by several manufacturers, and the market has consolidated. Cytiva’s Ettan Spot Picker is the system most often referred to in the literature and consumables for it are still sold. Bio-Rad’s EXQuest Spot Cutter no longer has its own product page. Other systems named in older literature include the Genomic Solutions ProPic, PerkinElmer ProXCISION, Bruker Proteineer DP and Herolab Spot Hunter. Because many labs still run one of these, SameSpots exports pick lists in the formats these instruments expect, plus a generic millimeter or pixel coordinate list for any other device.
Keeping keratin out
Keratin contamination from skin and hair is the leading cause of inconclusive mass spectrometry results from gel spots. It gets into the gel plug at every open-air step: cutting, transferring, destaining. Wear gloves, work in a laminar flow hood or a contained cutting environment, use fresh reagents and filtered tips, and keep tubes closed. Automated spot cutters with an enclosed working area exist largely to solve this problem.
In-gel digestion protocol
The values below are from the Rockefeller University Proteomics Resource Center’s published in-gel digestion protocol and are typical of the method. Trypsin is the usual enzyme because it cuts predictably after lysine and arginine, producing peptides of a size mass spectrometers handle well.
- Excise and wash. Cut the plug into roughly 1 mm pieces in a siliconized 0.5 ml tube. Destain with 50 mM ammonium bicarbonate in 50% acetonitrile until the stain is gone, then dehydrate the pieces in acetonitrile.
- Reduce and alkylate. Reduce with 10 mM DTT in 50 mM ammonium bicarbonate for 30 to 45 minutes at 55 °C. Alkylate with 55 mM iodoacetamide in 50 mM ammonium bicarbonate for 45 minutes at room temperature in the dark. This step is optional in the source protocol; it prevents disulfide bonds re-forming and gives cysteine a fixed modification the search engine can expect.
- Digest. Rehydrate the dried gel pieces in trypsin in 50 mM ammonium bicarbonate, pH 8, and incubate for 12 to 16 hours at 37 °C.
- Extract. Stop the digestion with 0.1% trifluoroacetic acid, extract peptides with 60% acetonitrile, 1% TFA, dry them down and resuspend in 0.1% TFA for MS.
Use an ultrasonic water bath to help extraction, and vortex between steps. Keep everything closed between steps; see keratin above.
Mass spectrometry: MALDI-TOF and LC-MS/MS
MALDI-TOF and peptide mass fingerprinting
The peptide mixture is spotted onto a target plate with a matrix, ionized by laser, and the masses of the intact peptides are measured. The list of peptide masses is the protein’s fingerprint, and it is matched against the masses predicted for every protein in a sequence database digested in silico. Peptide mass fingerprinting (PMF) is fast and cheap per sample and works well for a well-separated spot from an organism with a complete genome. It struggles when a spot contains more than one protein, when the genome is incomplete, or when the protein is small and yields few peptides.
MS/MS and LC-MS/MS
In tandem MS the instrument selects individual peptides and fragments them, and the fragment masses give the peptide sequence. MALDI-TOF/TOF does this from the target plate; LC-MS/MS separates the peptides by reversed-phase chromatography first and fragments them as they elute, with electrospray ionization. MS/MS identifies proteins from far fewer peptides than PMF, resolves mixtures, and can localize post-translational modifications. It is now the default for spot identification where the instrument is available.
Database searching
Both approaches end with a search of the measured masses against a protein sequence database. Mascot is the search engine most associated with 2D gel spot identification and is the format SameSpots imports directly; SEQUEST, MaxQuant and others are widely used for LC-MS/MS data. Each returns a ranked list of candidate proteins with a score and, for MS/MS, a list of matched peptides.
Read the result critically. A spot is a region of a gel, not a guarantee of one protein: in roughly 30% of spots the dominant protein accounts for less than 70% of the signal, according to Marcus, Lelong and Rabilloud. Where two proteins are identified in one spot, the expression change you saw on the gel may belong to either.
Identification failure was once common. A review in Clinical Proteomics notes that the historic rate of spot identification failure was estimated at up to 60% before improvements in gel image analysis. Better spot detection, better alignment and better pick-list accuracy are a large part of why that figure has fallen.
From analysis to pick list
In SameSpots the pick list is the end of the analysis rather than a separate job. Spots are ranked by the statistics, tagged, and exported with their coordinates in the format your picker expects or as a manual template. When the identifications come back from Mascot or a custom database, they are imported and attached to the matching spots, so the final report links each spot to its expression profile, its statistics and its identity.
Frequently asked questions
Q: What is a protein gel spot cutting system?
A: A robot that excises protein spots from a 2D gel at coordinates supplied by the analysis software, depositing each plug in a numbered microplate well for digestion and mass spectrometry. Pick heads are typically 1.5 mm or 3 mm.
Q: Can I cut spots from a 2D gel by hand?
A: Yes, for small numbers. Print the gel image at actual size with spots marked, place it under the gel on a light box, and cut with a clean tip or manual cutter. Keep keratin out and record which tube holds which spot.
Q: How are proteins identified from 2D gel spots?
A: The spot is digested in-gel, usually with trypsin, and the peptides are analyzed by MALDI-TOF (peptide mass fingerprinting) or by MS/MS, then matched against a sequence database with a search engine such as Mascot.
Q: What is peptide mass fingerprinting?
A: Identification of a protein from the masses of its tryptic peptides alone, without sequencing them. It is fast and works well for single-protein spots from organisms with complete genomes.
Q: Why do spot identifications fail?
A: Too little protein in the spot, more than one protein in the spot, keratin contamination, incomplete digestion, or an incomplete sequence database. Picking from a preparative gel and controlling contamination address the most common causes.
Q: Does SameSpots export pick lists?
A: Yes, in the formats used by spot-picking robots including the Cytiva Ettan Spot Picker, and as a generic coordinate list or manual template. Mass spectrometry identifications can be imported back from Mascot and custom databases.
References
1. The Rockefeller University Proteomics Resource Center. In-gel Digestion Protocol. https://www.rockefeller.edu/proteomics/gel-digestion-protocol/ (Source for: destain, reduction, alkylation, digestion and extraction reagents, concentrations, times and temperatures.)
2. Bio-Rad Laboratories. 2D Protein Spot Excision and Detection. https://www.bio-rad.com/en-us/applications-technologies/2d-protein-spot-excision-detection (Source for: keratin contamination as the leading cause of inconclusive mass spectrometry results.)
3. Magdeldin S, Enany S, Yoshida Y, et al. Basics and recent advances of two dimensional-polyacrylamide gel electrophoresis. Clinical Proteomics. 2014;11:16. https://doi.org/10.1186/1559-0275-11-16 (Source for: the historic spot identification failure rate estimated at up to 60% before improvements in image analysis.)
4. Marcus K, Lelong C, Rabilloud T. What room for two-dimensional gel-based proteomics in a shotgun proteomics world? Proteomes. 2020;8(3):17. https://doi.org/10.3390/proteomes8030017 (Source for: the dominant protein accounting for less than 70% of spot signal in about 30% of spots.)
From significant spot to pick list in one workflow
SameSpots ranks the spots that changed, exports the pick list in your picker’s format, and imports the identifications back onto the map. Request a trial and run it on your own gels.