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.