Home › Analytical Chem › Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa
Analytical Chem JoVE (Open Access) Citable · DOI

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

DOI: 10.3791/50635-v
What you'll learn
  • ✓ Prepare and optimize MALDI-TOF matrices for large protein analysis
  • ✓ Execute sample deposition and target preparation protocols
  • ✓ Acquire and interpret mass spectra for proteins >100 kDa
  • ✓ Troubleshoot matrix purity and sample crystallization issues
Protocol

Biopharma Insights Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Difficulty
intermediate
Total time
~3–4 hours per sample set (including matrix preparation, target cleaning, and data acquisition)

Steps

1
Recrystallize matrices to improve purity (optional)

Perform optional recrystallization of α-CHCA, DHB, or other MALDI matrices to remove contaminants and enhance ionization efficiency. This step improves data quality for sensitive analyses.

▶ 01:09
2
Clean MALDI stainless steel target plate

Thoroughly clean the MALDI target to remove residual salts, proteins, and contaminants. A clean target surface is essential for consistent crystallization and accurate mass measurement.

▶ 02:06
3
Prepare and deposit α-CHCA thin layer

Prepare a thin-layer α-CHCA matrix solution and deposit it evenly on the cleaned target. This thin-layer approach improves salt tolerance and crystal homogeneity.

▶ 02:49
4
Prepare matrix solutions for large proteins

Prepare working solutions of α-CHCA, DHB, and CHCA/DHB mixtures optimized for proteins >100 kDa. Different matrix compositions enhance ionization of large biomolecules.

▶ 03:18
5
Deposit sample and acquire MALDI spectra

Apply protein sample onto the prepared matrix layer and acquire MALDI-TOF mass spectra. Optimize laser power and acquisition parameters for large protein detection.

▶ 04:04
6
Analyze MALDI-TOF results for large proteins

Examine acquired mass spectra to determine accurate molecular weights and assess sample purity. Interpret peak patterns and mass accuracy for proteins exceeding 100 kDa.

▶ 05:17
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