2D Protein Spot Excision and Identification

Streamline 2D Protein Spot Excision and Identification with TotalLab’s Software

For many proteomics workflows, the ultimate goal of a 2D experiment is to identify differentially expressed proteins – whether it be for biomarker discovery, quality control, or antibody validation. This is achieved by mass spectrometric analysis of individual protein spots isolated and identified on a 2D gel. The spots of interest are excised from the gel and the protein is subjected to proteolytic treatment prior to identification by mass spectrometry. This section reviews the spot excision methods and protein identification tools available.

At TotalLab, we provide powerful tools to support this process: SameSpots, for 2D differential expression studies, and SpotMap 2D, for 2D Western blot-based host cell protein (HCP) coverage workflows.

Spot Excision: Precision Matters

Differentially expressed proteins identified by image analysis software such as SameSpots or SpotMap 2D can be marked on a printed image of the protein gel (see screenshot from our SameSpots software below), underlaid below the actual physical gel and the spots can then be manually and accurately cut from the gel.

SameSpots is also compatible with the majority of automated spot picking robots such as:

Exporting picking lists in their required file types to automate the process of protein retrieval from 2D SDS PAGE gels for proteomics based mass spectrometry and identification. Additionally our software also offers a generic file format with units in either mm or pixels which you can use in control files for other devices.

From 2D Gels to Mass Spec: A Smoother Workflow

After spot excision, proteins are typically de-stained and enzymatically digested (using something like trypsin for example) and can then be identified using a variety of techniques including peptide mass fingerprinting using matrix-assisted laser desorption ionization with time of flight mass spectrometry (MALDI — TOF MS), MALDI tandem MS using MALDI—TOF—TOF mass spectrometry, or by liquid chromatography tandem (LC) MS using reversed-phase chromatography coupled online to the mass spectrometer via an electrospray ionization source. In the first approach, proteins are identified by comparing the peptide masses against a protein sequence database. In the latter two approaches, proteins are identified using peptide masses and their MS-MS fragmentation patterns to search the protein database.

TotalLab software helps enable this workflow by allowing users to:

Identify and match protein spots across gels
Prioritize spots for excision based on expression differences
Document and export spot data for downstream workflows

Whether you’re working with Coomassie, Flamingo™, or fluorescent stains, SameSpots and SpotMap can detect and quantify spots with high sensitivity — even in complex serum or cell lysate samples.

SameSpots & SpotMap 2D: Your 2D Gel Partners

SameSpots: Designed for differential expression analysis using DIGE or Coomassie-stained gels.
SpotMap 2D: Built for 2D Western blot and HCP antibody validation workflows in GMP and 21 CFR Part 11 environments.

Both platforms support vendor-neutral imaging, multi-user collaboration, and audit-ready workflows — making them ideal for labs seeking high data quality and regulatory compliance.