NVoy Technology Facilitates Protein Mass Spectrometry

The generation of a sufficiently concentrated protein solution for                             
applications such as mass spectrometry can be problematic due to
protein aggregation and poor stability. NVoy technology can be
used to protect and stabilise proteins and is directly compatible with
mass spectrometry techniques such as Electrospray Ionisation
(ESI) and Matrix Assisted Laser Desorption Ionisation (MALDI).
This contrasts with many other commonly used stabilisers (see
Table 1) which are incompatible with either ESI or MALDI
techniques and cannot readily be removed effectively.

PROTOCOL

Aggregation and stability are very protein specific, but a general protocol is given below.
    1. Determine the starting protein concentration (using eg. Bradford assay, BCA assay, absorbance at 280nm).
    2. ESI & MALDI typically require a 5 μM solution of protein. This can be calculated using:
            [Protein] (mg/ml) = 5 (μM) x formula weight (Da) / 1000,000
    3. The presence of a tenfold excess, by mass, of NV10 should protect the target protein from aggregation during
        concentration. For example, 0.1 mg/ml NV10 will protect 0.01 mg/ml target protein.
    4. ESI and MALDI can tolerate up to 2.5 mg/ml NV10 in solution without causing interference.
    5. Each Stabil-P.A.C. tube contains 1.25 mg NV10 as a lyophilised powder.
    6. Add the protein solution to NV10 in Stabil-P.A.C. tubes to get the desired concentration, or make up a
        2.5 mg/ml stock of NV10 (1X stock) by adding 500 μL of buffer or distilled water to each Stabil-P.A.C. tube
        and add this stock to the protein solution.
    7. Concentrate the protein / NV10 solution to the desired protein concentration for mass spectrometry analysis.
    8. NV10 1X stock solution can be stored for 1 week at 4 oC or for longer term at -20 oC.
 

Troubleshooting

Buffers which are required for protein purification frequently interfere with mass spectrometry techniques. In such
cases the protein can still be purified, processed and concentrated in the presence of NV10 to prevent loss by
aggregation and non-specific binding and then the contaminating buffers can be removed using Zip Tip (Millipore)
purification immediately before sample application.

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Summary
Novexin’s NVoy technology enables the preparation of concentrated protein solutions
suitable for direct application in mass spectrometers using either electrospray ionisation
(ESI) or matrix assisted desorption ionisation (MALDI).