Electrospray ionization (ESI) is an ionization technique for small amounts of large and/or labile molecules such as peptides, proteins, organometallics, and polymers. The ESI source operates at atmospheric pressure. A sample solution is sprayed from a small tube into a strong electric field in the presence of a flow of warm nitrogen to assist desolvation. The droplets formed evaporate in a region maintained at a vacuum of several torr causing the charge to increase on the droplets. The multiply charged ions then enter the analyzer. The most obvious feature of an ESI spectrum is that the ions carry multiple charges, which reduces their mass-to-charge ratio compared to a singly charged species. This allows mass spectra to be obtained for large molecules. For example, apo-myoglobin with a molecular weight of 16,951.5 Da produces a series of ions with charge states from +8 to +27 with mass peaks from about 600 to 2000 Da.
We typically use 50/50 H2O/ACN as the mobile flow phase in ESI and samples are injected using a Rheodyne valve with a 10 microliter loop. Samples can also be introduced through direct infusion using a syringe pump. In addition, complex mixtures can be analyzed by coupling ESI MS with liquid chromatography (LC-MS).
Both positive and negative-ion spectra can be obtained. For positive-ion mode, 0.1% formic acid or acetic acid is usually added into the analyte solution to enhance protonation and increase sensitivity. For negative-ion mode, 0.3% NH4OH is usually added into the analyte solution to help deprotonation and increase sensitivity. For proteins, we typically use a concentration of 100 pmol/microliter in 50/50 H2O/ACN with 0.1% formic acid. Compounds such as porphyrins and other organometallic compounds are typically run in CHCl3 at a concentration of about 100 pmol/microliter.
Buffers such as phosphate, tris, and hepes cannot be used. Even trace levels of these interfere with the ESI process. Only volatile buffers such as ammonium acetate can be used. Excess Na+, K+, and detergents are very bad for ESI and frequently result in no data. Detergents (PEG's and PPG's) are especially bad because they work very well by ESI and suppress ionization of analytes. It is best not to add acid to the sample before submitting for ESI. We will dilute the sample to the proper concentration just before running the sample.
A Note about Proteins and Peptides: Proteins and peptides in solution should not be stored in glass vials. It is our experience that proteins will disappear irreversibly onto the glass. Samples in solution should be stored in Eppendorf-type centrifuge tubes. You should use the smallest tubes available (250 microliter) for very small amounts of sample.
Output is in the form of multiple charge series and transformed data.
Sample amounts needed for ESI
|Dry Sample (preferred)