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Inv e s t Igat Ing ga s Com p osIt Io n o n t r a ns p o rt a n d d e so lvat Io n o f H Ig H m/z s p eC I e s In t H e f I r s t vaC uum s tag e s o f a ma s s s p eC t rom e t e r
Iain Campuzano and Kevin Giles Waters Corporation, Manchester, UK
INT RODUCT ION
Here, we present a method that provides efficient desolvation
and effective transport of large biological ions from atmospheric
pressure into the vacuum system of a mass spectrometer, through
modification of the atmospheric pressure gas (cone gas) composi-
tion. No other source modifications were carried out in this work.
Significant enhancements of the transmission and desolvation of
large multiply-charged protein ions were achieved by changing the
cone gas from nitrogen to a significantly heavier polyatomic gas.
Nanoelectrospray is an ionization technique that efficiently gener-
ates large biological gas-phase ions. Transfer of non-covalently
associated protein-protein complexes from solution to the gas phase
generally results in the formation of ions possessing relatively few
charges, and consequently m/z values are often above 10,000. This
depends on the size of the protein complex under investigation.
Biological samples analyzed under non-denaturing aqueous
conditions are heavily solvated, resulting in non-gaussian mass
spectral peak shape. An efficient desolvation process results in
an accurately measured intact mass of the complex, due to the
stripping of non-specific solution adducts.
Figure 1. Schematic of the Z-Spray Source Block and point of introduction of cone gas.
EX PERIMENTAL
The instrument used in these studies was a SYNAPT™ HDMS™
System, which has a hybrid quadrupole/IMS/oa-ToF geometry. Briefly,
samples were introduced by a borosilcate glass nanoelectrospray
spray tip and sampled into the vacuum system through a Z-Spray™
source. The ions passed through a quadrupole mass filter to the IMS
section of the instrument. This section is comprised of three travelling
wave (T-Wave™) ion guides. The trap T-Wave accumulates ions, while
the previous mobility separation occurs. These ions were released
in a packet into the IMS T-Wave in which the mobility separation was
performed. The transfer T-Wave was used to deliver the mobility
separated ions into the oa-ToF analyzer.
SAMPLES AND GASES
Alcohol dehydrogenase (147.5 kDa), GroEL-SR (400 kDa, single
ring), Proteasome-S (700 kDa), and GroEL (800 kDa) were all
buffer exchanged into an aqueous solution of 100 mM ammonium
acetate, to a final working protein concentration of 1.5 µM.
Sulphur hexafluoride (SF6) was obtained from BOC Gases LTD.
Octafluoropropane was obtained from F2-Chemicals, UK.
SF6 and Octafluoropropane were introduced as cone gas through
the sheath cone assembly, as shown in Figure 1. The flow rate was
controlled by means of a software controlled Bronkhorst gas flow
controller (standard). The flow rate was varied from 0 L/hour to
100 L/hour to investigate optimal conditions for GroEL (800 kDa)
detection and transmission.
RESULTS
To enable accurate comparisons of GroEL transmission when
assessing SF6 and Octafluoropropane, the deconvolution algorithm
Maximum Entropy 1 was used to generate an intensity value for
the entire GroEL multiply-charged envelope. This figure was then
plotted against the cone gas flow rate, as shown in Figure 2. SF6,
when used as a cone gas, “cools” the large ions more efficiently
than Octafluoropropane. The multiply-charged GroEL ions were not
detectable when Nitrogen, Argon, or Xenon (data not shown) was
used as a cone gas.
Large multiply-charged ions possess a large energy spread within
the mass spectrometer. The kinetic energy spread reduced, so the
ions could be focused and ultimately detected. Polyatomic gases,
such as SF6 and Octaflouropropane, “cool” or “thermalize” large
ions, as shown in Figures 3 and 4, which resulted in enhanced detec-
tion. Polyatomic gases possess far more degrees-of-freedom than
monoatomic gasses, such as Argon or Xenon. For example, SF6 (Mw
146) and Octafluorpropane (Mr 188) possess 21 and 33 degrees-
of-freedom (3N) respectively, as opposed to the
3 degrees-of-freedom of Xenon (Mr 131).
Figure 2. Investigating the effect of cone gas SF6, Octafluoropropane, and Argon indi-cated flow rates (L/hour) on the transmission of intact GroEL (800 kDa).
Figure 3. Multiply charged GroEL spectra obtained at optimal flow rates of Octafluorpropane (85 L/hour) and SF6 (60 L/hour).
Figure 4. TOF-MS data acquisition of Yeast ADH, GroEL-SR, and Rabbit Proteasome-S, using SF6 as a cone gas.
30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
S1
0
1
2
3
4
5
6
SF6
Octafluoropropane
Argon
m/z2500 5000 7500 10000 12500 15000 17500 20000 22500 25000 27500 30000
%
0
100
%
0
10011452
11290
11133
10978
11620
11793
11972
11782
11611
11444
11281
89103843
11959
12142
12333
12526
Sulphurhexafluoride 50 L/hour
Octoflouropropane 8.5 L/hour
m/z2500 5000 7500 10000 12500 15000 17500 20000 22500 25000 27500 30000
%
0
100
%
0
100
%
0
1005678
5467
3081
2843
5273
5905
6152
8520
8343
8174
38221806
8903
8923
9109
9335
11980
11590
11412
11217
12042
12203
12255
12405
Rabbit Proteasome-S
720 kDa
GroEL-Single Ring
400 kDa
Yeast ADH
Tetramer 147.5 kDa
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
Waters is a registered trademark of Waters Corporation. The Science of What’s Possible, ZSpray, SYNAPT, HDMS, and T-Wave are trademarks of Waters Corporation. All other trademarks are property of their respective owners.
©2008 Waters Corporation Produced in the U.S.A. July 2008 720002744EN AG-PDF
CONCLUSIONS
Polyatomic gases used as a cone gas dramatically improved
transmission and desolvation of large multiply charged,
high m/z ions without the need for any source modification.
Improved transmission and efficient desolvation of large
multiply-charged ions allowed for accurate mass measurement
of large protein/protein noncovalent complexes.