BioFiles Volume 5, Number 2 — Protein Characterization & Detection

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MALDI Mass Spectrometry

 


Introduction

MALDI (matrix-assisted laser desorption/ionization) expands the application of mass spectrometry (MS) toward the analysis of high molecular weight, non-volatile and thermally labile compounds, such as intact proteins and oligonucleotides, and is a major technique in proteomics research. MALDI-MS also requires relatively little sample preparation and is more amenable to topological imaging compared to other forms of MS ionization.

The MALDI technique involves mixing the sample with a matrix substance and crystallizing the mixture on a MALDI sample plate to improve crystal uniformity using a variety of ways to achieve crystallization. The crystallized sample/matrix mixture is irradiated by a laser light source, usually with an ultraviolet spectral wavelength. As the matrix absorbs the light energy, it evaporates into the gas phase, resulting in an indirect ionization of the sample molecules. Although there have been experiments with direct MS from solid supports and 2D-gels in order to simplify MALDI by eliminating the samplematrix mixing step, using a chemical matrix is still the most common approach.


MALDI Matrices Quality

A typical MALDI matrix substance is an aromatic acid with a chromophore that absorbs strongly at the wavelength of the incident laser. Choosing a suitable matrix of high quality is key to the success of a MALDI-MS experiment. Organic impurities can lead to extraneous peaks, especially in the low mass range. Trace levels of ions, especially Na+ and K+, form adducts with sample molecules. These adducts differ in mass according to the number of positive ions and complicate the MS spectrum. Because of the stringent purity requirements for both organic impurities and inorganic ions, many MALDI users have resorted to repurifying commercially available but impure matrix substances.

The ever-increasing sensitivity of MALDI-MS instruments and an ongoing trend of decreasing sample quantities (e.g., proteins from excised spots from 2D-gels) require high purity and high quality MALDI reagents and matrices. Sigma® Life Science's Ultra pure MALDI matrices include the most commonly used matrix substances with strict, application-based specifications which include:

  • Minimum 99.5% purity
  • Trace cation impurity limits, typically ≤1 mg/kg (≤1 ppm)
  • Appearance and solubility requirements

Ultra pure matrix substances are extensively purified to meet these specifications and support the most demanding MS applications.

Figure 1. Ultra pure MALDI Matrix, 10 X 10 mg package size.


Excellent Performance of Ultra Pure MALDI Matrix Substances

One of the most important aspects of the Ultra pure MALDI matrix substances from Sigma is their ability to dissolve rapidly and completely; a brief vortex mixing is typically sufficient. Actual performance of the Ultra pure MALDI substances in situ was demonstrated using a test mixture of five ProteoMass™ peptide standards (see Figures 2 and 3). MALDI-MS experiments were performed on a Shimadzu™ Kratos PC Axima CFR V2.4.1 in reflectron mode. The comparison of MALDI-MS spectra using Ultra pure α-cyano-4-hydroxycinnamic acid (HCCA, Product No. 39468) versus reagent grade HCCA is shown in Figures 2a/b. Note that the use of the Ultra pure HCCA matrix resulted in significantly higher peak resolution. Even when the peptide standard sample was diluted 10-fold (Figure 3a/3b), the use of Ultra pure HCCA provides a spectrum with clear resolution (Figure 3a) comparable to the spectra using the more concentrated sample ( Figure 2 ). In contrast, the reagent grade HCCA did not yield a suitable spectrum with the diluted peptide standard (Figure 3b).

 


Figure 2a. HCCA Ultra pure grade (Product No. 39468).




Figure 2b. HCCA reagent grade.

Figure 2. Comparison of MALDI-MS spectra using Ultra pure and reagent grade HCCA as matrices. Sample: 0.7 μL ProteoMass peptide mix [bradykinin (1.5 μM), angiotensin II (1.0 μM), P14R (0.5 μM), ACTH (1.0 μM), insulin chain B (2.0 μM)] mixed with 0.7 μL HCCA matrix in ACN/0.1%TFA. Instrument: Shimadzu® Kratos Axima CFR, refl ectron mode, laser power 85%.
 
Figure 3a. HCCA Ultra pure grade (Product No. 39468).




Figure 3b. HCCA reagent grade.

Figure 3. Comparison of MALDI-MS spectra using Ultra pure and reagent grade HCCA as matrices with 10-fold diluted peptide sample. Sample: 0.7 µL ProteoMass peptide mix [bradykinin (150 nM), angiotensin II (100 nM), P14R (50 nM), ACTH (100 nM), insulin chain B (200 nM)] mixed with 0.7 µL HCCA matrix in ACN/0.1%TFA. Instrument: Shimadzu Kratos Axima CFR, refl ectron mode, laser power 85%.

 


The Perfect MALDI Companion: High Sensitivity LUCY® Fluorescent Stains

The excellent performance of the Ultra pure MALDI matrix substances with dilute samples or very small volume sample volumes, where high sensitivity is required, is of great benefit when analyzing peptides from enzymatic digests separated by 1D- or 2D-gels. In this case, however, not only the MALDI matrix must be of high purity, but the dyes used to stain the gels prior to excising the spots must allow the visualization of low abundance proteins. LUCY fluorescent dyes from Sigma Life Science deliver the ability to detect low abundance proteins (see Figure 4) for downstream MALDI-MS analyses. A 100 ng band of β-galactosidase was excised from a 1D-gel which was stained with LUCY 506 solution (Product No. 68721). After tryptic digestion and peptide extraction using the Trypsin Profile IGD Kit (Product No. PP0100), MALDI-MS was performed by mixing peptides with Ultra pure HCCA matrix (Product No. 39468). The protein could be identified by database analysis and peptide mass fingerprint from the resulting spectrum.


Figure 4. MALDI-MS peptide mass fingerprint after in-gel-digest.
100 ng of E. coli β-galactosidase, separated by SDS-PAGE, was stained
with LUCY 506 after band excision and trypsin digestion. Extracted peptides
were crystallized with HCCA and measured on a Shimadzu Kratos CFR
MALDI-MS instrument in reflectron mode.


Conclusion

Sigma offers a complete line of products that meet the sensitivity demands of modern MS analysis, including MALDI-MS:

  • Ultra pure matrix substances with extremely low levels of both organic impurities and inorganic ions.
  • LUCY fluorescent dyes improve visualization of peptides in 1D- and 2D-gels compared to competitive dyes.
  • The ProteoMass line of peptide standards is ideal for troubleshooting and calibrating sensitive MALDI-MS instruments.

MALDI-MS Matrices

Lucy Stains

ProteoMass MALDI-MS Calibration Kits and Standards

ProteoMass MALDI-MS Peptide Standards

MALDI Validation Sets

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