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OVERVIEW Here, we investigate a data independent

acquisition strategy aimed at intact protein mixtures analysis (i.e. “top-down”).

Following the first stage of separation (liquid chromatography) and ionization by ESI, multiple charge states of intact protein ions are presented to the collision cell (CID), without any precursor ion selection.

The CID collision potential is scanned, generating 2D patterns of product ion spectra versus collision potential.

The patterns can be used for characterization, identification and quantitation of protein mixtures.

DATA INDEPENDENT CHARACTERIZATION OF PROTEINS FROM 2D CID FINGERPRINTS GENERATED THROUGH COLLISION ENERGY SCANNING

Boris Kozlov, Jeff Brown, John Hoyes. Waters Corporation, UK

METHODS LC-CID-MS with range of collision potentials

For the LC-MS experiments, with scanning of the collision potential, we used an unmodified Xevo G2 XS Q-TOF (Waters) with TOF-MS resolving power at 30,000 (FWHM). This instrument has the option of recording of up to 32 functions, which can be chosen to run at different collision energies. The quadrupole was operated as an ion guide to allow all ions through to the collision cell. Ten MS functions were programmed with the following set of collision potentials: 5, 10, 15, 20, 30, 40, 60, 80, 100, and 140 V (Uc in figure 1). The scan time was 0.3 seconds for each function at each collision potential. The collision cell was pressurized with 20 microbar of argon. LC (Acquity UPLC, Protein BEH C4 column, 300A, 1.7um, 2.1x50 mm) solvent flow was 100 uL/min with acetonitrile concentration changed from 20 to 40% in 40 minutes. Direct infusion (single protein 2D-patterns)

Two dimensional CID patterns for individual proteins were obtained from the Citius TOF MS (Leco) in direct infusion experiments at 5 µL/min continuous sample flow. Proteins were ionized by ESI. The applied collision potential (defining the collision energy) was increased in 5 volt steps from 5 to 200 volts. The accumulation time for each collision energy spectrum was approximately 1 minute. The pressure inside the collision cell was 20 microbar of nitrogen. Resolving power of this instrument was 100,000 FWHM in high resolution mode and 5,000 FWHM in survey mode.

RESULTS

CONCLUSIONS

Fast scanning of the collision potential adds a new dimension of differentiation to LC-MS-MS analysis of proteins.

The method potentially allows the identification of proteins by prior knowledge of their product ion potential appearance func t ion . Fur thermore , improved quantification of proteins within complex mixtures is expected.

Figure 3. LC-MS heat-map (with zoomed central area) of intact protein mixture, with a low collision potential of 5 V (very little fragmentation).

Figure 4. 2D fragmentation pattern for cytochrome c obtained in direct infusion experiments. Top—overview. Bottom—zoom in m/z range 720—780 Th. Peak groups are marked with values of their charge state.

Figure 5. 2D fragmentation pattern for monoclonal antibody ions

Figure 6. 2D fragmentation pattern for bovine serum albumin ions. Inserts show zoom of different m/z ranges.

Figure 9. Bovine insulin. Evolu-tion of peaks in molecular ion cluster (Charge +5)

DISCUSSION

LC-MS-MS analysis of mixture of intact proteins can be performed with scanning of collision potential in real time during single LC run.

We obtained collision potential dependence mass spectra (2D-patterns) of fragments for some proteins.

All protein ions in current experiments are multiply charged and are detected between m/z 500 and 3000 .

With no collision energy applied mainly adducts instead of molecular ions are detected. To maximize molecular ion signal we applied collision potentials between 10 and 50 V.

With rise of collision energy some loss of charge leads to arising protein ions with lower charge state that are not recorded at zero collision energy.

Fragmentation potential of proteins depends on their charge state, rising nearly proportional to m/z.

Fragments appear in order of decreasing charge state when collision potential is increased.

In current experiments most of multiply charged “fragments of interest” are registered at about 25 – 100 V of collision potential. There may be a slight trend of lower averaged appearance potential for smaller proteins.

At high collision potentials (100 V and higher) we see a nearly homogeneous “carpet” of singly charged ions over a wide mass range.

The most informative pattern was seen for cytochrome-c (12 kDa) as compared with bovine insulin (6 kDa) or BSA (60 kDa). Low molecular weight (6 kDa) protein has a relatively low charge state of molecular ion (+4 -to +5), so there is low probability of breaking onto two multiply charged parts.

Figure 7. Some fragments (cytochrome C) versus collision potential (UC). For example, 0.05M+1 is the singly charged fragment ion at (m/z 617) with 1+ charge.

Figure 1. Ion optics of the Xevo G2 XS Q-TOF. The collision po-tential is applied at the entrance of collision cell.

Figure 2. 3D-array of LC-CID-MS data. Each plane shows 2D data for LC-MS at different collision potential (Uc) Right vertical plane shows cut of the array for chromatographic peak of cytochrome-c. Left vertical plane Shows TICs

Figure 8. Bovine Insulin fragmentation pattern. Some peak groups are marked with values of their charge state.