Electrospray ionization (ESI) is an ionization technique for small amounts of large and/or labile molecules such as peptide's, proteins, organometallics, and polymers. The ESI source operates at atmospheric pressure, so a quadrupole analyzer, which does not employ high voltages, is easier to interface to the ESI source. A solution of the sample is sprayed though a needle at potential of 3.5 kV. The voltage on the needle causes the spray to be charged as it is nebulized. The droplets evaporate in a region maintained at a vacuum of several torr causing the charge to increase on the droplets. Rapid evaporation of the droplets allows protonated analyte molecules to be released into the gas phase. The multiply charged ions then enter the analyzer. In principle, all molecules that can be charged are amenable to ESI/MS analysis. ESI is a soft ionization technique and is particularly useful for obtaining the molecular weight of analytes that would normally exceed the normal mass range of sector and quadrupole instruments. 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 amu produces a series of ions with charge states from +8 to +27 with mass peaks from about 600 to 2000 amu. Hence we can see analytes with a mass up to 100,000 on a quadrupole instrument with a mass range of only 4000 amu.
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. Many solvents can be used in ESI. We frequently use CHCl3 and THF. Samples can also be introduced via a Harvard Model 22 syringe pump using gas tight syringes. Although 100% water can be used in ESI, better sensitivity is obtained with some organic solvent present in the water. Even 5-10% of MeOH or AcN increases the stability of the nebulization process.
For proteins we typically use a concentration of 10 pmol/microliter in 50/50 H2O/AcN with 0.1% formic acid. If the sample is not ionic to begin with, then some source of protons must be added to the sample. Anionic compounds are run in basic solution using 0.3% NH4Acetate. Compounds such as porphyrins and other organometallic compounds are typically run in CHCl3 (sometimes with 0.1% formic acid added to promote cationization) 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 (PEGs and PPGs) are especially bad because they work very well by ESI.
It is best not to add acid or solvent to the sample before submitting for ESI or MALDI.
I will dilute the sample to the proper concentration just before running the sample.
Samples should be submitted in Eppendorf-type tubes 0.5ml or 1.5ml. You can obtain some
from me if you need them.
I generally require a minimum of 1 mg of sample for analysis. Note I will return samples back to you when completed.
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.
Wednesday, 02 June 2010
By Frank Antolasic