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Surface modified nanoliter spray probe improves mass spectrometry sensitivity

Hello, everyone. This week, I will share an article published on J. am Soc. Article on mass spectrum, surface modified nano electrospray needs improved sensitivity for native mass spectrum [1] 。 The corresponding author of this article is Professor Michael T. Marty from the University of Arizona in the United States.

Non denaturing mass spectrometry (NMS) and charge detection mass spectrometry (CD-MS) have become multifunctional tools for characterizing various protein and polymer complexes. Both usually use borosilicate needles for Nano Spray Ionization (nESI). However, because the protein is usually positively charged at neutral pH, it may be adsorbed on the negatively charged glass nESI needle surface, thus reducing the sensitivity and affecting the data analysis.In order to improve the sensitivity of NMS and CD-MS, the author modified the surface of nsei needle with inert surface modifier. By covalently connecting polyethylene glycol (PEG) to the surface of silanol, the glass surface is passivated to reduce non-specific adsorption.

First, in order to determine whether surface modification can improve the sensitivity of mass spectrometry, the author’s team used peg coated glass nESI needles to detect two non-specific glass adsorbed proteins:bovine serum albumin (BSA) and lysozyme. The results showed that compared with the control group, the signal intensity of BSA and lysozyme increased by about 2 times (Fig. 1). PEG coating significantly improved the MS sensitivity of nESI needle to standard protein.

Figure 1 (A) BSA raw mass spectra of uncoated control needles and (b) peg coated needles showed signal strength. (C) The signal intensity of lysozyme and (d) BSA PEG coating (light blue) and control (gray) nESI needle.

Next, the authors used CD-MS equipped with PEG surface coated nESI needle to detect the intact adenovirus (AAV) capsid. The results showed that the total number of ions collected by PEG modified needle was more than 8 times higher than that of the control group without modified needle at lower concentration (Fig. 2). Compared with general CD-MS detection, the sample concentration detected by CD-MS with improved needle is lower and the collection time is shortened.

Figure 2 CD-MS analysis of AAV2 capsid. (A) Control group; (B) Peg coated needle. (C) The total number of individual ions collected from a 5-minute CD-MS acquisition of an empty AAV2 capsid.


Next, the author studied the influence of the size and geometric shape of nESI needle tip on the experimental results. The experiment found that although the improved needle significantly improved the signal intensity at a lower concentration, there was a great difference between the needles. The authors’ team hypothesized that the deviation in signal intensity was caused by the difference in tip diameter of the manually trimmed nESI needle. In order to minimize the changes of nESI needle tip size and geometry, the author developed a needle puller program to repeat production with 2 μ NESI needle with a diameter of M. It was found that PEG modified 2 μ M needle can significantly improve the detection signal strength, and the difference between each operation is small. Compared with manually trimmed needles, 2 μ The signal of M needle increases more. zero point one μ M nESI pin and 2 μ The signal intensity of the protein detected by m-needle and m-needle was similar (Fig. 3).

Based on the above results, the author speculates that 2 μ The reason why the signal value detected by M pin is higher may be 2 μ The taper of M needle is shorter. A shorter taper may produce a higher coating density near the tip of the needle. The needle cut manually has a long taper, which damages the PEG coating near the tip during the drawing process, so the detected signal value is low. And 0 one μ M and 2 μ The taper of M needle tip is relatively short, and the coating may be intact when it is close to the surface of the needle tip, so the signal intensity detected by the two is similar.

Figure 3 With 2 μ M (left) and 0.1 μ M (right) tip diameter of PEG coated (light blue) and uncoated control (gray) nESI needle signal intensity of the most abundant charge state of BSA.

Through the above experiments, the authors have confirmed that PEG modified nESI can improve the sensitivity of NMS and cd-ms. Next, the author makes an in-depth study of its mechanism. First, the authors tested whether the increase in sensitivity was due to the reduction of nonspecific adsorption on glass. The authors used two coatings with different chemical properties:PEG and pfdcs to modify the needle. Both of them can reduce the non-specific adsorption of protein and theoretically improve the sensitivity of mass spectrometry. However, it was found that only peg coated needles could improve the signal strength.

Then, the author used two kinds of needles to detect the ubiquitin signal value. Ubiquitin does not adsorb with glass under neutral conditions. Theoretically, there is no statistical difference between the two signal values, but the results show that compared with pfdcs modified needle, the signal value detected by PEG modified needle group is 3 times higher. It is concluded that the peg coated needle is not a mechanism to improve the mass spectrum signal value by reducing the non-specific adsorption between protein and glass.

Finally, the author studied the capillary effect of the surface modified needle and found that the unmodified borosilicate capillary had the strongest capillary effect, the peg capillary had medium strength capillary effect, and the pfdcs capillary had almost no capillary effect (FIG. 4A). Then, the flow rates of different nESI needles were studied under static conditions without fluid pumping or pressure application (Fig. 4b). The results showed that the flow rate of PEG modified nESI needle was the highest, while there was no significant difference between pfdcs modified and control nESI needle. The authors assume that the increase in sensitivity may be caused by the increase in the flow rate of nESI needle. Due to the high capillary force in the traditional needle, the liquid will tightly adhere to the glass, reducing the liquid flow at a given ESI voltage. The PEG modification reduces the capillary resistance, which may increase the liquid flowing to the tip, thereby increasing the signal. Although pfdcs modified needle has low capillarity, its flow rate is small, which may be due to the need for a certain strength of capillarity to obtain the best flow velocity. The author will further explore this hypothesis in future experiments.

Photos of capillary action of ESI needle. (B) Flow rates of pfdcs modified (dark blue), PEG modified (light blue), and unmodified (gray) needles.

to make a long story short,The author proved that PEG modified nESI needle increased the mass spectrum signal strength and sensitivity of many analytes, and showed a relatively fast and low-cost method that can improve the sensitivity of difficult analytes at low concentrations. The authors speculate that surface modification can improve the sensitivity of mass spectrometry by increasing the flow rate at the tip of nESI needle, but this speculation still needs further proof.

[1]Kostelic MM, Hsieh CC, Sanders HM, Zak CK, Ryan JP, Baker ES, Aspinwall CA, Marty MT. Surface Modified Nano-Electrospray Needles Improve Sensitivity for Native Mass Spectrometry. J Am Soc Mass Spectrom. 2022 Jun 1; 33(6):1031-1037. doi:10.1021/jasms. 2c00087. Epub 2022 May 19. PMID:35588532.