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Electrospray Ionization
Mass Spectrometry: Electrospray
Ionization
Electrospray ionization mass spectrometry (ESMS) is
carried out on a Micromass Q-Tof mass spectrometer on
samples with molecular weights from 100 to well over 100,000
(More
Information). The technique is sensitive in that it
usually can be carried out on low pmol amounts of sample.
Samples should be free of detergents, polyethylene glycol
type compounds, and salts (e.g., as little as 5 mM
NaCl precludes analysis). Upon request, samples that contain
salts or other non-volatile reagents may be subjected to a
reverse-phase desalting on a ZipTip®,
however, this procedure may (particularly in the case of
proteins) lead to significant loss of sample. Volatile
buffers (such as 1-10 mM ammonium carbonate or acetate) are
acceptable and, if necessary, samples may be injected that
contain up to 10% (v/v) glycerol. Many different volatile
solvent systems can be used (e.g., water or
water/organic combinations utilizing acetonitrile,
isopropanol, or chloroform/methanol). Unless requested
otherwise, all samples will be introduced as a solution in
50% acetonitrile, 0.2% formic acid and will be run with
positive ion detection. Since the analysis will fail if the
sample is not soluble in this buffer, it is important that
the solubility of the sample in 50% acetonitrile, 0.2%
formic acid be confirmed prior to sample submission.
Typically, 10 µl of a 1-10 µM protein solution is
needed for an analysis. The following provides a brief
description of several types of commonly requested analyses.
In addition, many other types of samples (e.g.,
carbohydrates, lipids, and polymers) may also be analyzed on
the Q-Tof. It is important to note also that the average
mass accuracies given below are estimated and that the mass
error on any given sample may be either higher or lower than
these estimates. Monoisotopic masses should be used for
molecules with mass less than 3 kDa while average masses
should be used for larger molecules.
Nominal Mass Determinations on Synthetic Molecules Above
150 Da
This type of analysis is extremely useful for rapid
monitoring of chemical syntheses where an average mass error
of ± 0.2 amu (400 ppm or ± 0.04% for 500 Da) is
sufficient to determine if the synthesis is indeed
proceeding as planned. This level of mass accuracy can be
achieved routinely on the Q-Tof with external calibration
and without having to constantly monitor instrument
calibration.
Exact Mass Determinations on Synthetic Chemical
Intermediates (150-1000 Da)
To reach the extremely high level of mass accuracy
required to confirm the elemental composition of the
products of chemical syntheses requires the use of internal
calibrants. To avoid signal suppression, the concentration
of these internal calibrants have to be carefully matched to
that of the sample. The average mass accuracies that may be
achieved with this approach are ± 0.002 Da over the
mass range 150-400 and 5 ppm (± 0.0005%) over the mass
range extending from 400-1000)
High Mass Accuracy Determinations on Peptides (100-2500
Da)
Frequent monitoring of the external calibration of the
Q-Tof allows the average mass error with external
calibration to be decreased to less than ±0.05 amu (50
ppm or ±0.005% for 1000 Da).
ESMS Analysis of Oligonucleotides
ESMS spectra of oligonucleotides are recorded in negative
ion mode. To increase sensitivity and mass accuracy the
samples should be free of sodium and potassium counterions.
There are many published methods to remove or minimize the
effect of these counterions. These include organic
precipitation, RP-HPLC, ZipTip clean-up (which also rellies
on a reverse phase support), or carrying out the ESMS in the
presence of triethylamine acetate, piperidine, or imidazole
buffers. We use the latter two methods: ZipTip clean-up
and/or one of the three buffers mentioned. The longest
oligonucleotide we have analyzed was a 39-mer. ESMS spectra
of longer oligos have been reported in the literature. We
request that a minimum of a 100 pmol be submitted. Any
unused sample can be returned if requested beforehand.
ESMS Analysis of Proteins
For large molecular weight biomolecules (e.g.,
proteins), it is important to recognize that the measured
mass is the average mass and that the peak envelope extends
over many individual masses. For example, a protein with a
mass of 10 kDa. will have a peak envelope that is
approximately 20 mass units wide (counting all isotope
containing peaks with intensities greater than 1% of the
most abundant peak, for a detailed discussion see Anal.
Chem. 55,353-356 (1983)). As the size of the molecule
increases, the peak envelope gets wider such that the
envelope for a 100 kDa protein should contain 57 molecular
ion peaks. In instruments that cannot resolve these
individual isotopic peaks, the width of this isotopic
envelope will determine what mass difference is needed
between two molecules in order to detect each molecule. As a
general guide, one should be able to theoretically detect
two components present in equal amounts if their molecular
weight divided by their mass difference is less than
approximately 1,000. Hence, if the samples are very clean
(i.e., no counterions or adducts which would create
'tails' on the mass spectral peaks) it may be possible to
detect a 50,000 Da species in the presence of an equivalent
concentration of a 50,050 dalton species.
ESMS spectra are almost always recorded in positive ion
mode. The mass spectrum from a protein contains a series of
peaks produced from the protein by adding a successive
number of protons. Because mass spectrometers record
mass-to-charge ratio, electrospray analyses on proteins
typically produce a spectrum in the mass range of 500-2000.
This spectrum containing multiply charged peaks is processed
to produce a peak on a molecular mass scale by either
transforming manually identified multiply charged peaks
(adjacent peaks can only differ by one charge) or by MaxEnt
transformation which only requires an accurate peak width as
input. The MaxEnt transformation produces a mock spectrum
which can be compared to the raw data to determine the
quality of the transformation. The average, expected mass
error is about ± 0.01%. (More
Information).
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