Method for improved signal-to-noise for multiply charged ions

A method of improving the signal-to-noise using first and second mass spectrometers in tandem, with an ion detector and data system coupled to the second mass spectrometer, comprising selecting precursor ions with the first mass spectrometer, at least some of the parent ions being multiply charged, colliding or reacting the precursor ions in an intermediate chamber so that multiply charged parent ions produce product ions which have at least one fewer charge than the multiply charged precursor ions, and using the second mass spectrometer or the ion detector and data system to allow only those ions which have an m/z value higher than the multiply charged precursor ions to be recorded for analysis by the ion detector and data system, so that only a signal due to multiply charged precursor ions is obtained in said data system.

FIELD OF THE INVENTION 
This invention relates to a method for using a triple quadrupole mass 
spectrometer, or another type of tandem mass spectrometer, to improve the 
signal-to-noise ratio in the mass spectrum of a sample. The invention has 
particular application to a mass spectrum produced by electrospray or ion 
spray. In particular, the method relates to improving the signal-to-noise 
("S/N") ratio for multiply charged ions (which are ions containing two or 
more charges) in the presence of unwanted signal background which consists 
mainly of singly charged ions. 
BACKGROUND OF THE INVENTION 
In producing a mass spectrum for a sample, particularly when methods such 
as electrospray or ion spray are used, there is commonly unwanted 
background noise. It is always desirable to have the signal level 
relatively high compared with the background, in order to be able to 
distinguish the signal. Many approaches, some elaborate and expensive, 
have been used to achieve this objective. 
The inventors have found that when the sample is used to produce multiply 
charged ions (as commonly occurs in electrospray, ion spray and related 
techniques), the background consists mainly of singly charged ions. In 
this situation, using the techniques described below, it is possible to 
reduce the unwanted background considerably (i.e. to improve the S/N 
ratio). 
BRIEF SUMMARY OF THE INVENTION 
In a preferred aspect the present invention provides a method of improving 
the signal-to-noise using first and second mass spectrometers in tandem, 
with an ion detector and data system coupled to the second mass 
spectrometer, comprising selecting precursor ions with the first mass 
spectrometer, at least some of the precursor ions being multiply charged 
and having at least a predetermined number of charges, colliding or 
reacting the precursor ions in an intermediate chamber so that multiply 
charged precursor ions produce product ions which have at least one fewer 
charge than the multiply charged precursor ions with the predetermined 
number of charges, and using the second mass spectrometer or the ion 
detector and data system to allow only those ions which have an m/z value 
higher than the multiply charged precursor ions having at least the 
predetermined number of charges to be recorded for analysis by the ion 
detector and data system, so that only a signal due to multiply charged 
precursor ions having at least the predetermined number of charges is 
obtained in said data system. 
Further objects and advantages of the invention will appear from the 
following description, taken together with the accompanying drawings.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
Reference is first made to FIG. 1, which shows diagrammatically a 
conventional triple quadrupole mass spectrometer system 10 with which the 
invention may be used. (Other mass spectrometer systems may also be used.) 
As shown, the system 10 includes a sample source 12, which is typically an 
electrospray source, an ion spray source, or another ion source which 
produces a multiply charged ion stream 14. The ions 14 are injected 
through an orifice 16 in an orifice plate 18, through a vacuum chamber 20, 
and through an orifice 22 in a skimmer 24, into a first quadrupole Q1 
located in a vacuum chamber 26. Quadrupole Q1, which has both AC and DC 
applied to it, acts as a resolving mass spectrometer, transmitting ions 
having a selected mass to charge (m/z) ratio while rejecting other ions. 
Ions which are transmitted through quadrupole Q1 pass through an orifice 28 
in an interface plate 30 into another vacuum chamber 32 containing a 
quadrupole Q2 located in a "can" 34 and arranged to operate as a collision 
cell. Collision gas at a desired pressure is supplied from collision gas 
source 36. Normally quadrupole Q2 has RF-only applied thereto (without 
resolving DC), so that quadrupole Q2 can contain and transmit ions having 
a wide range of m/z values. 
In quadrupole Q2, parent ions fragment by collisions with the collision gas 
to produce product or daughter ions. Ions emerging from quadrupole Q2 pass 
through an orifice 38 in an interface plate 40 into a quadrupole Q3 which 
has both AC and DC applied thereto (again from a source not shown), to act 
as a resolving quadrupole, transmitting only ions having a selected m/z 
ratio. The RF and DC applied to Q3 are normally scanned to produce a mass 
spectrum detected at detector 42 and read by a computer data system 44. 
In the method of the invention, advantage is taken of the fact that a 
multiply charged ion can be fragmented, e.g. in a collision cell, to form 
at least some product or daughter ions which are less charged, and which 
have a higher m/z value than the parent ion. An example of such ions are 
doubly charged peptide ions. 
Therefore, if it is desired to detect the presence of a small amount of 
this doubly charged ion, in the presence of higher levels of singly 
charged ions (which may be present due to other components in the sample, 
or due to ions from the solvent used in the sample source 12), then as 
mass spectrometer Q1 is scanned through a mass range of interest, the ions 
transmitted through Q1 are fragmented in collision cell Q2, and mass 
spectrometer Q3, or the data system 44 which is connected to the detector 
42, is set to transmit or record only those ions which have a greater m/z 
value than that which is transmitted by the first mass spectrometer Q1. 
Ideally, the computer and data system 44 will record a signal which is the 
sum of the signal-from all ions greater in mass than the precursor or 
parent ion transmitted through Q1, in order to achieve high sensitivity. 
The resulting mass spectrum will show mass peaks corresponding only to 
those ions which have two or more charges, since all ions with only one 
charge will fragment only to m/z values lower than that of the precursor 
or parent ion. 
This method may be performed using a triple quadrupole, with reference to 
the standard quadrupole stability diagram as shown in FIG. 2, which plots 
a on the vertical axis and q on the horizontal axis, where 
##EQU1## 
where e is the electron charge, V is the RF amplitude, U is the DC 
amplitude, m is the mass of the ion of interest, .OMEGA. is the RF 
frequency (radians/second), and r.sub.0 is the inscribed radius of the 
space within the rods. 
When the quadrupole is operated in the resolving mode, the operating line 
usually is set to pass through the tip 46 of the stability diagram, where 
q=0.706. Ions having q=0.706 are therefore transmitted, while ions having 
masses such that their q's are less than or greater than 0.706 are 
rejected to the rods. 
When the quadrupole is operated in RF-only or total ion mode, without DC 
(so that the operating line is on the q axis), ions having q.gtoreq.0.908 
are rejected to the rods. 
Therefore, in one embodiment of the invention, and with reference to FIG. 
1, Q1 is operated in a resolving mode; Q2 is operated as a conventional 
collision cell, and Q3 (which normally would be operated in a resolving 
mode) is instead operated in a total ion mode (i.e. as an RF-only 
quadrupole), at an RF level which corresponds to more than 9/7 that of the 
RF level in Q1 (i.e. at a "q" value greater than 0.908 for masses which 
were transmitted through Q1). In this mode, Q3 operates as a high pass 
filter, transmitting only ions which have a greater m/z ratio than those 
which are transmitted by Q1 (since Q1 is as mentioned operated near the 
tip of the stability diagram, at about q=0.706). Thus, only fragment ions 
formed in Q2 which are greater in m/z than the precursor or parent ion 
will be transmitted through Q3. As the RF (and DC) levels are scanned in 
Q1 (to transmit ions of different masses), the RF level in Q3 is 
correspondingly scanned to maintain its amplitude at more than 9/7 that of 
the RF level in Q1. 
An advantage of operating Q3 in a total ion mode is that high sensitivity 
is achieved, since there are no transmission losses associated with 
resolving in Q3, and there is no loss in efficiency which would be 
associated with scanning Q3 over a selected mass range for each Q1 m/z 
value. By operating Q3 in an RF-only mode, at a fixed ratio (greater than 
9/7) compared to Q1, the detector 42 receives only ion signal from ions 
which have two or more charges (since ions with more than two charges also 
fragment to produce ions higher in m/z value than the precursor, so that 
doubly, triply, quadruply, etc. charged ions would all be observed in the 
spectrum, but singly charged ions would not). 
The efficiency of the process described depends on how efficiently each 
species is fragmented to ions higher in m/z than the precursor. This will 
vary from one ion species to another, but it is generally true that all 
multiply charged ions will form at least some higher m/z fragments. Since 
it is generally true that higher m/z precursors require higher collision 
energy in order to fragment, the collision energy should be scanned over a 
defined range as Q1 is scanned through a selected mass range. For example, 
the collision energy (per single charge) could be scanned upwardly from 30 
eV to 100 eV as the precursor or parent ion m/z is scanned from 400 amu to 
2000 amu. It is noted that if the collision energy is too low, then the 
ions in Q2 may not have sufficient energy to fragment, while if the 
collision energy is too high, then the initial product or daughter ions 
may fragment further before they leave the collision cell Q2, resulting in 
predominantly low m/z fragments, which would defeat the purpose. The 
optimum relationship between m/z and collision energy can be determined 
empirically. Even though an empirically determined relationship for some 
classes of compounds will not be optimum for all compounds, it will be 
preferable to fixing the collision energy for a wide m/z range. 
The ideal ratio (R) for the ratio of Q3/Q1 m/z values (RF voltages) is 
slightly greater than 0.908/0.706=1.286, i.e. preferably R.about.1.3. This 
will eliminate transmission of the unfragmented precursor or parent ions 
through Q3, but will allow transmission of any lower charge state ions 
which were even only slightly greater in m/z than the precursor ion. 
Another way of accomplishing the above-described method is to use the 
second mass spectrometer Q3 in a mass resolving mode, and only record ion 
signal from those fragments above the precursor or parent ion mass. While 
this is not desirable for a triple quadrupole (because for each m/z 
transmitted by Q1, Q3 would have to be scanned over a significant mass 
range in order to detect all of the higher m/z ions, and this would be 
very slow), it is very practical with a tandem mass spectrometer 50 of the 
kind shown in FIG. 3, where primed reference numerals indicate parts 
corresponding to those of FIG. 1. 
In the mass spectrometer system shown in FIG. 3, the third quadrupole Q3 
has been replaced by a time-of-flight (TOF) mass spectrometer 52. In the 
system 50, TOF spectrometer 52 simultaneously records all of the fragment 
ions, separated in time. Thus, to record only those ions higher in m/z 
than the precursor, the computer data system 44' is programmed to record 
or extract only those ions which are higher in m/z than the precursor, and 
to produce a spectrum which consists of the sum of intensities of all 
these ions, via the m/z of the precursor. This method also results in 
detecting only those ions which form fragments with an m/z value higher 
than that of the precursor. The same method can be used with a first mass 
analyzer Q1, collision cell Q2 (or other fragmentation means), and a 
second mass analyzer consisting of a magnetic sector mass spectrometer and 
a spatial detector (for example an array detector). In this case, the 
fragment ions are dispersed in space, and the array detector can be 
adjusted to detect or record only ions which are greater in m/z than the 
precursor. 
FIG. 4A shows a mass spectrum (obtained with Q1 of a triple quadrupole such 
as system 10) of myoglobin, a protein which forms a series of multiply 
charged peaks 62. The peaks 62 due to myoglobin appear above a background 
of signal which is of unknown origin but which may be composed of singly 
charged background ions. Only the peaks from charge state 21 and above are 
easily distinguishable above the background. 
In FIG. 4B, the described method has been applied, by fragmenting ions in 
Q2, and operating Q3 in an RF-only mode (i.e. a total ion mode) at an m/z 
value which is 400 amu above that of Q1. Therefore, for all values of m/z 
less than 1400, the ratio of Q3/Q1 RF voltages (m/z values under normal 
calibration conditions) is greater than 9/7, and only ions which fragment 
to an m/z greater than that of the precursor mass will be transmitted 
through Q3 and detected. Thus all charge states below 12+ are detected due 
to their fragmentation to ions having higher m/z values. Charge states 12+ 
and 11+ are transmitted through both Q1 and Q3. 
It is clear that the signal-to-noise ratio is better in FIG. 4B than in 
FIG. 4A, due to removal of a significant portion of the background by the 
described method. Charge states 24+, 23+ and 22+ are clearly visible in 
FIG. 4B but not in FIG. 4A. While for this experiment Q1 and Q3 were 
scanned with a fixed mass value difference of 400 amu, it would be 
preferable to scan Q3/Q1 in a fixed ratio of greater than 9/7. This was 
not performed here because the software did not provide that scan method. 
If it is desired to detect only ions which have more than two charges, then 
Q3 can be scanned in a ratio of greater than 2.times.9/7=2.58 to Q1 (i.e. 
Q3/Q1&gt;2.58). This will record only signals from ions which fragment to 
singly charged ions at greater than the m/z of the doubly charged 
precursor. In general, ions with greater than n charges can be detected 
while discriminating against ions with a lower number of charges, by 
operating Q3 at a value of 9n/7 of Q1. However as n increases, the 
probability of forming a fragment which is 9n/7 higher than the precursor 
decreases. 
A second example is shown in FIGS. 5A and 5B. These show spectra 70, 72 
which were obtained using the system 50 of FIG. 3, i.e. a quadrupole Q1, a 
collision cell Q2, and a TOF mass spectrometer 52. The spectrum 70 shows a 
mixture of peptides from a chemical digestive cytochrome c, a protein. 
Spectrum 70 was obtained from the TOF analyzer 52 without any 
fragmentation, and with quadrupole Q1 operated in a conventional RF-only 
mode. Many peaks appear in the spectrum, some associated with the peptides 
in the sample, others possibly associated with solvent or buffer or 
contaminant ions. The peptide ions of interest are usually doubly or 
triply charged. In fact, it is well known that doubly charged peptide ions 
fragment in the most predictable fashion to provide sequence information. 
Therefore it is often desired to detect which of the ions in the spectrum 
70 are doubly or triply charged, so that they can be subjected to MS/MS. 
While the isotope pattern is often sufficient to distinguish the doubly 
and triply charged ions (doubly charged ions have isotopic peaks spaced 
0.5 amu apart, and triply charged species 0.33 amu apart), in many cases 
the surrounding peaks prevent easy identification in this manner. For 
example, the first isotopic peak may be completely masked by the peak from 
a singly charged ion, so that it cannot be determined which is the first 
peak in the isotopic cluster. If the wrong peak is selected, the 
calculated mass value could be in error by 1 Da. 
In mass spectrum 72 of FIG. 5B, obtained from the same sample by scanning 
with Q1 in a mass resolving mode, (RF and DC applied), fragmenting the 
ions in collision cell Q2, and recording only those ions in the TOF 
spectrum which have m/z values greater than the precursor (the value 
transmitted through Q1), many of the smaller peaks, as well as the singly 
charged peaks, from FIG. 4A are absent. Only the doubly and triply charged 
ions are evident. This demonstrates the benefit of the method in improving 
the signal-to-noise ratio for multiply charged ions. 
The method described can be employed with any combination of mass 
analyzers, in which the ions are fragmented between the analyzers and the 
second analyzer is used to detect or distinguish only those species which 
are greater than a threshold, where the threshold indicates a lower limit 
on the charge state. In addition, the method can be used to distinguish 
which ions in a complex spectra are singly charged, or lower than a 
certain charge state, by comparing the described scan (showing only 
multiply charged ions) to the full scan to show only the lower charge 
state ions. 
In a related method, higher charge state ions can be reduced in charge 
state by allowing them to react in the collision cell Q2 with neutral 
species which will acquire one or more charges from the ion. For example, 
a low concentration of water vapor, or methanol vapor, or another 
gas-phase base, may be added to the collision gas. At low entrance energy, 
multiply charged ions may react by transferring one or more charges to the 
neutral molecule. This effectively increases the m/z of the ion. Thus, in 
the same way as described above, the second mass analyzer (Q3 or the TOF 
for example) can be used to detect only those ions which react to generate 
products which are higher in m/z than the precursor. 
Another method of accomplishing the described effect is to operate the 
collision cell at an RF level which is slightly higher than 9/7 of the RF 
level of Q1. Only fragment ions formed in the collision cell through 
reaction or fragmentation which are higher in m/z than the precursor ion 
will then be stable and will be transmitted to the TOF analyzer or to Q3. 
The precursor plus lower m/z fragments will be unstable and thus will be 
rejected by the collision cell. This removes the requirement for using the 
computer data system 44 to sum all of the ions greater than the precursor 
m/z value. Since the energy of precursor ions entering the collision cell 
is high, the precursor will penetrate to a sufficient distance to 
accomplish a few collisions before being rejected from the cell due to 
instability. Therefore, multiply charged ions which fragment or react 
close to the entrance of the collision cell, and which form ions higher in 
m/z value than the precursor ion, will be detected by measuring the total 
ion current from the TOF analyzer, or by measuring the ion current after 
the collision cell Q2. 
While preferred embodiments of the invention have been described, it will 
be realized that various changes may be made within the scope of the 
invention.