B American Society for Mass Spectrometry, 2013
J. Am. Soc. Mass Spectrom. (2014) 25:286Y292 DOI: 10.1007/s13361-013-0763-1
RESEARCH ARTICLE
A New Splitting Method for Both Analytical and Preparative LC/MS Yi Cai, Daniel Adams, Hao Chen Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA
Abstract. This paper presents a novel splitting method for liquid chromatography/ mass spectrometry (LC/MS) application, which allows fast MS detection of LCMS separated analytes and subsequent online analyte collection. In this approach, a PEEK capillary tube with a micro-orifice drilled on the tube side wall is used to connect with LC column. A small portion of LC eluent emerging from the orifice can be directly ionized by desorption electrospray ionization (DESI) with LC negligible time delay (6~10 ms) while the remaining analytes exiting the tube outlet can be collected. The DESI-MS analysis of eluted compounds shows eluent narrow peaks and high sensitivity because of the extremely small dead volume of the orifice used for LC eluent splitting (as low as 4 nL) and the freedom to choose favorable DESI spray solvent. In addition, online derivatization using reactive DESI is possible for supercharging proteins and for enhancing their signals without introducing extra dead volume. Unlike UV detector used in traditional preparative LC experiments, this method is applicable to compounds without chromophores (e.g., saccharides) due to the use of MS detector. Furthermore, this splitting method well suits monolithic column-based ultra-fast LC separation at a high elution flow rate of 4 mL/min. Key words: LC/MS, Eluent splitting, DESI, Protein supercharging, Ultra-fast LC
DESI spray
Received: 23 August 2013/Revised: 18 September 2013/Accepted: 28 September 2013/Published online: 20 November 2013
Introduction
S
plitting the eluent in liquid chromatography/mass spectrometry (LC/MS) experiments is often necessary under the circumstance when the mobile phase flow rate is too high for MS ionization. For instance, electrospray ionization (ESI), a common ionization method for coupling LC with MS, requires an optimal sample infusion rate at μL/min level while the mobile phase flow rate for chromatographic separation using regular analytical LC columns is in the range of 1~2 mL/min. Higher flow rates (up to 9 mL/ min) are in need for ultra-fast LC separation using monolithic columns [1, 2]. In addition, post-column splitting is also needed when the remaining portion of LC eluent after MS detection needs to be collected for preparative purpose. Typically, postcolumn splitting of LC eluent can be made simply using a Tee splitter in which one of the split streams goes to MS for detection and the other one can go to waste or to a second Electronic supplementary material The online version of this article (doi:10.1007/s13361-013-0763-1) contains supplementary material, which is available to authorized users. Correspondence to: Hao Chen; e-mail: [email protected]
detector [3, 4]. However, the connection capillary bridging the Tee and MS would introduce dead volume and could cause peak broadening [5, 6]. The addition of a splitter could also increase the dead volume, the column back pressure and the cost of experiment. In addition, there is a time delay for MS detection because of the significantly reduced flow rate of the split eluent flowing into the MS source (the flow rate can be dropped by as much as 100 times). Therefore, it is practically difficult to collect purified analytes in the other “waste” stream (because the analyte in the “waste” stream could flow out even earlier than being detected by MS). Thus, a new splitting method with fast MS detection and reduced dead volume is desired, to assist both online detection and online sample collection. Desorption electrospray ionization (DESI) originally developed by Cooks has been introduced for direct sample ionization with little or no sample preparation [7–15]. Besides analysis of solid samples, in our and other laboratories, DESI has been extended for direct liquid sample analysis [16–29]. In this study, we present a new splitting interface for LC/MS applications based on fast DESI ionization capability, which allows MS monitoring of the LC eluent with minimal time delay (in milliseconds) and online collection of the major portion of separated analytes for preparation purpose.
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
287
alcohol (m-NBA, ≥ 99.5 %), N-acetyl-D-glucosamine ( ≥ 99 %), maltohexaose, sulfadiazine (HPLC grade), sulfamerazine (HPLC grade, ≥ 98.8 %), and sulfaquinoxaline (HPLC grade, ≥ 96 %) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid was purchased from Fisher Chemicals (Pittsburgh, PA, USA). HPLC-grade methanol was also purchased from Fisher Chemicals. HPLC-grade acetonitrile was purchased from EMD Chemicals Inc. (Billerica, MA, USA).
In this approach, a PEEK capillary tube carries a microdrilled orifice on its side wall (Scheme 1) and is connected with the LC column outlet. When the LC eluent flows through the PEEK tube, a small aliquot of eluent emerges out of the orifice and can be directly sampled and ionized by DESI while the remaining analytes exiting from the tube outlet can be collected and further characterized. Advantages of such a new splitting method in combination with DESI-MS detection are severalfold. First, it minimizes the dead volume (as low as 4 nL) involved in LC eluent splitting because there is no need to use a connection tubing to bridge the splitter and MS. Thus, the occurrence of peak broadening can be avoided and high detection sensitivity can be achieved. Second, due to the prompt DESI detection of separated analytes, it is convenient to collect purified analytes from the PEEK tube outlet. This capability expands the applications of preparative liquid chromatography. Traditionally, such a collection is facilitated by using a UV detector placed between the LC column and the collection sample reservoir. In this case, MS detection allows detecting and collecting UV-transparent compounds following LC/MS analysis. Third, reactive DESI [30, 31] can be used for online derivatization, for example, for supercharging proteins without introducing an extra dead volume. Last, the application of this proposed splitting method with DESI-MS detection to monolithic column-based separation, for which a high mobile-phase flow rate is needed for ultra-fast elution, was demonstrated to be viable.
LC Separation Conditions A commercial PerkinElmer HPLC system (Perkin Elmer, Shelton, CT, USA) was used throughout the experiments. For the protein mixture separation, a Waters XB Bridge TM 300 C4 column (4.6 mm × 150 mm) was employed with a trifluoroacetic acid (TFA)-containing mobile phase of ACN:H2O:TFA (30:70:0.1 by volume) being used. The mobile phase flow rate used was 1.0 mL/min. For the saccharide mixture separation, an Agilent (Santa Clara, CA, USA) ZORBAX ODS C18 column (4.6 mm × 250 mm) was adopted with 0.1 % FA in H2O used as the mobile phase at the flow rate of 1.0 mL/min. For the mixture of the sulfonamides, a monolithic C18 column (Phenomenex (Torrance, CA, USA), Onyx, 100 × 4.6 mm) was chosen. A gradient elution program from 100 % A down to 90 % A in 4 min was used (mobile phase composition A: water and B: ACN) with a elution flow rate of 4 mL/min. A 20 μL injection loop was used for sample loading.
Experimental Chemicals
DESI-MS Detection
Insulin (from bovine pancreas, HPLC grade), ubiquitin (from bovine erythrocytes), trifluoroacetic acid (TFA), 3-nitrobenzyl
A home-built liquid DESI ion source with a Thermo Finnigan (San Jose, CA, USA) LCQ DECA ion trap mass
N2
DESI spray probe
LC column
. ...
Sample mixture
DESI spray 530 µm
. PEEK tubing
MS . ... .. Orifice (i.d. 350 µm)
.
510 µm
Outlet
PEEK tubing
Scheme 1. Apparatus of a new LC/MS post-column splitting method in which a DESI spray probe is used to ionize analytes of interest in the eluent emerging out of a micro-drilled orifice from the PEEK tube. The remaining portion of eluent exiting the PEEK tube outlet can be collected for preparative LC. Inset illustrates the zoomed-in PEEK tube with the orifice
288
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
spectrometer (Scheme 1) was used throughout the experiments. A short piece of PEEK tube (i.d. 510 μm; wall thickness: 530 μm; length: 3 cm) with a micro-drilled orifice (i.d. 350 μm) was connected to the LC column. The orifice was located in the tube 2 cm downstream from the LC column and the tube outlet was slightly bent downward to facilitate sample collection. The sample eluent flowing out of the orifice underwent interactions with the charged microdroplets generated from DESI spray for ionization. Unless specified, the spray solvent for DESI was CH3OH/H2O/HOAc (50:50:1 by volume) and injected at 10 μL/min with 5 kV applied to the spray solvent. The DESI spray probe was placed above the orifice and the distance between the probe and orifice was about 1–2 mm. The orifice was placed approximately 1 cm away from the MS inlet.
demonstration of the feasibility of this proposed splitting method in conjunction with DESI detection for LC/MS coupling. For separation, a reversed phase C4 column with mobile phase of ACN:H2O:TFA (30:70:0.1 by volume) was employed. TFA is often used as a mobile phase modifier to promote protein separation as TFA not only can adjust pH but also serves as an ion-pairing agent. But TFA is not an “ESI-friendly” reagent because of its signal suppression effect [32]. With the eluent from LC column flowing through the PEEK tube at a flow rate of 1.0 mL/min, part of the eluent (30 % in this prototype experiment) emerging from the PEEK orifice was ionized by DESI. As shown in the extracted ion chromatograms (EICs, Figure 1a and b), two proteins were well separated and the peak widths for insulin and ubiquitin are approximately 0.7 and 1.0 min, respectively. The resulting DESI-MS spectra (Figure 1c and d) also clearly display the ionized individual proteins with multiple charge distribution. In addition, it takes only 10 ms for the sample eluent to go through the micro-drilled orifice, which is calculated based on the flow rate of eluent through
Results and Discussion In this study, a protein mixture of insulin and ubiquitin (100 μM each) was first chosen as a test sample for LC/DESI-MS
(a)
100
0 100
LC/Reactive DESI-MS, with m-NBA +4
+5
EIC of insulin NL: 1.76E6
(a’) EIC of insulin
100
NL: 2.83E7
(b) EIC of ubiquitin
0 100
+6
(b’) EIC of ubiquitin
Relative Abundance
NL: 2.69E6
0 0.0
0.5
1.0
+7
NL: 2.02E7
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0 0.0
5.5
0.5
1.0
1.5
2.0
2.5
100
+4
(c) Insulin
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Time (min)
Time (min) +5
(c’) Insulin
100
NL : 2.15E6
NL : 2.42E5
+4
50
50
0
100
600
800
1000
1200
(d) Ubiquitin
+6
+3
+5 1400
1600
1800
+3 2000
0
600
+6
+7
+5
+9
800
1000
1400
1200
1400
m/z
1600
1800
2000
0
1600
1800
+6
+10 +11 +12 +13 600
800
2000
+8
+9 50
+10 +11 +12 600
1200
+7
NL: 1.30E6
+8
50
1000
(d’) Ubiquitin 100
NL : 2.26E5
0
800
+5 1000
1200
1400
1600
1800
2000
m/z
Figure 1. EICs of (a) insulin (+4 ion) and (b) ubiquitin (+6 ion) from LC/DESI-MS analysis and the corresponding DESI-MS spectra of (c) insulin and (d) ubiquitin. EICs of (a′) insulin (+5 ion at m/z = 1148) and (b′) ubiquitin (+7 ion at m/z = 1224) from LC/ reactive DESI-MS analysis where m-NBA was doped into the DESI spray solvent and the corresponding reactive DESI-MS spectra of (c′) insulin and (d′) ubiquitin
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
the orifice (300 μL/min) and the calculated orifice dead volume of 50 nL (i.d. 350 μm; tube wall thickness: 530 μm). Therefore, DESI-MS used in this experiment virtually offers a “near-real time” monitoring of LC eluent stream in the PEEK tube. For comparison purpose, electrosonic spray ionization [33] (ESSI, a variant form of ESI that employs an ESI source with supersonic nebulization gas for better desolvation effect) was also used to detect the LC separated analytes. In the experiment, the LC eluent flow rate was reduced to 10 μL/min (an optimized flow rate for ESSI ionization) using a commercial splitter (ASI adjustable commercial splitter), for the reason that the high flow of LC eluent at 1.0 mL/min would flood the ESSI ion source. LC separation experimental conditions were kept the same as those used in LC/DESI-MS analysis mentioned above. As revealed by the resulting EIC spectra (Figure 1S-a and S-b, Supporting Information) from ESSI-MS detection, the two EIC peaks of the proteins are much wider than those recorded using LC/ DESI-MS, each of which lasts about 4 min (Figure 1S-a and S-b, Supporting Information). It is probably caused by the increased dead volume from the traditional splitting method used for ESSI ionization. Presumably because of peak broadening (or a more severe TFA ion suppression effect), the EIC intensities from ESSI-MS detection (Figure 1S-a and S-b, Supporting Information) are lower than those from DESI-MS detection (Figure 1a and b). In addition to splitting LC eluent without introducing significant dead volume to allow fast MS monitoring, our method has an additional benefit for enabling online derivatization using reactive DESI. Reactive DESI [30, 31], in which a chosen chemical reagent is doped with the DESI spray solvent, is applicable in this LC/DESI-MS method, for supercharging proteins and for enhancing their signals. Such an online supercharging experiment would not introduce an extra dead volume as the supercharging occurs during DESI ionization. The charge enhancement for proteins by supercharging is of great value for further structural analysis via top-down approaches for increasing the fragmentation efficiency [34, 35]. It is known that 3-nitrobenzyl alcohol (m-NBA) is an effective supercharging reagent [36]. Indeed, as m-NBA was doped in the DESI spray, the maximum charge state of insulin shifted from +5 to +6 and the charge with highest abundance shifted from +4 to +5 (Figure 1c′) compared with the regular DESI data (Figure 1c). Likewise, the maximum charge state of ubiquitin shifted from +12 to +13 and the charge with highest abundance shifted from +6 to +7 (Figure 1d′). Interestingly, the signals of resulting protein ions in this LC/reactive DESI-MS (Figure 1a′ and b′) are significantly enhanced in comparison to those of regular DESI (Figure 1a and b) and ESSI ionization (Figure 1S-a and S-b, Supporting Information). This phenomenon is in agreement with our previous observation [37]. Presumably, m-NBA reduces TFA dissociation [38] so that the decreased concentration of trifluoroacetate anions reduces their ion pairing interaction with protein cationic sites, a mechanism responsible for signal suppression of TFA.
289
Besides the analytical strength, another important feature of this method is to collect isolated samples following LC/ MS analysis. Indeed, with the aid of online DESI monitoring as mentioned above, the major portion of insulin and ubiquitin exiting from the PEEK tube outlet (70 %) were collected. Re-analysis of the two collected samples by ESSIMS gave rise to the spectra of isolated insulin and ubiquitin (Figure 2S-a and S-b, Supporting Information), showing no cross-talking and confirming that the two samples were completely separated and successfully collected. Conventionally, in the preparative LC experiment, the collection of LC-separated eluent is often facilitated by placing a UV detector between the LC separation column and the sample collection reservoir. However, such an approach is limited to compounds with chromophores. For those without chromophores, derivatization is in need, which is time-consuming and troublesome. By using our DESI method, this problem can be solved when MS servers as a detector in our method. As a demonstration, a saccharide mixture consisting of Nacetyl-D-glucosamine (NAG) and maltohexaose (2.5 mM each) with no or weak UV absorption was chosen as a test sample and a C18 column was used for separation. As shown in the recorded EIC spectra, NAG and maltohexaose were well separated, and each of the saccharide peak width is about 0.6 min (Figure 2a and b). Note that although there is no organic solvent used in the mobile phase (only 0.1 % FA in H2O was used as the mobile phase), the saccharides can still be well detected by DESI-MS. This is ascribed to the freedom of choosing favorable DESI spray solvent of CH3OH/H2O/HOAc (50:50:1 by volume) for sample ionization. This phenomenon agrees with previous reports [18, 24]. Following the LC/DESI-MS analysis, the remaining saccharides were online collected and re-tested with MS, which again shows the full separation of two saccharides (Figure 2c and d). Interestingly, the eluent splitting ratio provided by the PEEK tube with the orifice can be adjusted. Simply by reducing the orifice i.d. from 350 μm down to 100 μm, the splitting ratio of 3:7 can be reduced to 4:96 for which more sample can be collected after LC separation. Experiment was further conducted to evaluate the performance of the PEEK tube carrying the 100 μm i.d. orifice. In this case, the time for eluent going through the orifice channel was only 6 ms (the split flow rate: 40 μL/min; orifice dead volume: 4 nL). By still using a mixture of insulin and ubiquitin as a test sample, the EIC spectra acquired show good protein separation (Figure 3S-a and S-b, Supporting Information). In comparison to Figure 1a and b, the signals of both proteins are even higher, probably because a lesser amount of TFA is involved in the sample being ionized. Also, the proteins after LC separation were collected, and re-analysis of the collected samples show clean spectra (Figure 3S-c and S-d, Supporting Information). We quantified the yield of collection using UV spectroscopy, which shows 94.2 % ± 0.6 % average yield for insulin and 94.4 % ± 1.8 % average yield for ubiquitin (see discussion in the Supporting
290
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
[NAG + H]+
[NAG + H]+
[2NAG + H]+
(c) Collected N-acetyl-D-glucosamine
100
100
(a)LC/DESI-MS EIC of N-acetyl-D-glucosamine NL: 2.70E6 50
Relative Abundance
[NAG - H2O + H]+
[3NAG + H]+
[4NAG + H]+
0
0
200
[Maltohexaose + H]+
100
(b) LC/DESI-MS
100
300
400
500
600
700
(d) Collected maltohexaose
800
900
[Maltohexaose +
1000
H]+
EIC of maltohexaose NL: 1.75E6
50
[Maltohexaose + Na]+
223 0 0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
200
5.0
300
400
500
600
700
800
900
1000
m/z
Time (min)
Figure 2. EICs of (a) NAG and (b) maltohexaose from LC/DESI-MS analysis. MS spectra of (c) the collected NAG and (d) the collected maltohexaose. The peak at m/z 223 in (d) came from solvent background
Information). These yields are fairly close to the theoretical value of 96 % based on the splitting ratio, suggestion the
feasibility and potential of this approach for sample purification.
[Sulfamerazine + H]+
100
a) LC/DESI-MS [Sulfaquinoxaline + H]+
Relative Abundance
[Sulfadiazine + H]+
0 0.0
0.5
1.0
1.5
2.0
2.5
Sulfadiazine
Sulfaquinoxaline
100
0.5
3.5
Sulfamerazine
b) LC/UV
0.0
3.0
1.0
1.5
2.0
2.5
3.0
3.5
Time (min) Figure 3. EIC spectra of a mixture of sulfadiazine, sulfamerazine, and sulfaquinoxaline (a) from LC/DESI-MS analysis and (b) from LC/UV analysis. The LC column used was Onyx C18 monolithic column and the PEEK tube with a 100 μm i.d. orifice was utilized. The first peak in UV spectrum might be from gas bubble introduced during sample loading
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
Besides the applications to LC separation using regular analytical columns, we also examined the compatibility of our splitting method to monolithic column-based ultra-fast LC separation. Monolithic column is a kind of “single-piece column” that has been developed for fast high-throughput analysis [39]. It allows performing high-throughput analysis with good separation performance close to that of ultra-high performance LC [2, 39, 40].The key factor in such an LC experiment is that an extremely high flow rate (up to 9 mL/min) can be used with monolithic columns without causing significantly high back pressure. Thus, the separation can be completed in a short period of time. In our experiment, a mixture of sulfonamides, including sulfadiazine, sulfamerazine, and sulfaquinoxaline (100 μM each), drugs commonly used for eliminating bacteria and treating urinary tract infections, was chosen as a test sample for demonstration of the application of our splitting method to the monolithic column-based ultra-fast LC separation. As shown in EIC spectrum recorded by DESI-MS (Figure 3a), the sulfadiazine, sulfamerazine, and sulfaquinoxaline were all separated and eluted within 3 min. In comparison, when a regular reversedphase C18 column with an elution flow rate of 1 mL/min is used for the separation of the same mixture, the total elution time is 11 min. (Figure 5S, Supporting Information). This result shows that the high flow rate does help fast elution and separation. We also recorded the chromatogram using an UV detector (detection wavelength: 254 nm) for comparison (Figure 3b). The DESIMS-recorded chromatogram has slightly better resolution than that recorded by UV detector. For instance, there is about 10 % overlap of the sulfadiazine and sulfamerazine peaks in Figure 3b, while the two peaks are well resolved in the DESIMS recorded chromatogram. This might be also due to the overall small dead volume involved in our splitting method. However, attempt to split the high flow eluent of 4 mL/min down to 10 μL/min for ESSI-MS detection by the commercial splitter caused column failure because of high back pressure that exceeds the pressure tolerance of the monolithic column used. In contrast, in our split method in conjunction with DESI-MS detection, there is no back pressure issue as the PEEK tube used for splitting has an opening that is not blocked or linked with another device. In addition, for showing the real world sample applications, we also used this LC/DESI-MS for analysis of αlactalbumin tryptic digest. Beside identifying 11 separated peptides in the digest, we were able to successfully isolate one peptide containing an intramolecular disulfide bond resulting from protein disulfide bond scrambling during trypsin digestion at basic pH (this peptide is of interest because it is useful for studying how pH affects disulfide bond scrambling in proteins, an important issue in proteomics. See detailed discussion in Supporting Information).
Conclusions This paper reports a new and versatile splitting method for LC/MS coupling, as an additional example of combining LC with MS using ambient ionization methods [22, 37, 41, 42].
291
Premium analytical performance of DESI and successful sample purification with high collection yield was demonstrated. Narrow peak width, high sensitivity, no back pressure issue, and low cost are among the striking features of the presented methodology. Owing to the capability of DESI for ionizing both small organic molecules and high-mass proteins, this method would have wide applications in bioanalysis.
Acknowledgments The authors acknowledge support for this work by NSF Career Award (CHE-1149367).
References 1. Su, Z.H., Zou, G.A., Preiss, A., Zhang, H.W., Zou, Z.M.: Online identification of the antioxidant constituents of traditional Chinese medicine formula Chaihu-Shu-Gan-San by LC-LTQ Orbitrap mass spectrometry and microplate spectrophotometer. J. Pharmaceut. Biomed. Anal. 53, 454–461 (2010) 2. Sangoi, M.S., Todeschini, V., Steppe, M.: Fesoterodine stress degradation behavior by liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry. Talanta 84, 1068–1079 (2011) 3. Schiavo, S., Ebbel, E., Sharma, S., Matson, W., Kristal, B.S., Hersch, S., Vouros, P.: Metabolite identification using a nanoelectrospray LC-ECarray-MS integrated system. Anal. Chem. 80, 5912–5923 (2008) 4. Andrews, C.L., Li, F., Yang, E., Yu, C.-P., Vouros, P.: Incorporation of a nanosplitter interface into an LC-MS-RD system to facilitate drug metabolism studies. J. Mass Spectrom. 41, 43–49 (2006) 5. Camenzuli, M., Goodie, T.A., Bassanese, D.N., Francis, P.S., Barnett, N.W., Ritchie, H., LaDine, J., Shalliker, R.A., Conlan, X.A.: The use of parallel segmented outlet flow columns for enhanced mass spectral sensitivity at high chromatographic flow rates. Rapid Commun. Mass Spectrom. 26, 943–949 (2012) 6. Andrews, C.L., Yu, C.-P., Yang, E., Vouros, P.: Improved liquid chromatography-Mass spectrometry performance in quantitative analysis using a nanosplitter interface. J. Chromatogr. A 1053, 151–159 (2004) 7. Harris, G.A., Galhena, A.S., Fernandez, F.M.: Ambient sampling/ ionization mass spectrometry: applications and current trends. Anal. Chem. 83, 4508–4538 (2011) 8. Douglass, K.A., Venter, A.R.: Protein analysis by desorption electrospray ionization mass spectrometry and related methods. J. Mass Spectrom. 48, 553–560 (2013) 9. Zhang, S., Shin, Y.-S., Mayer, R., Basile, F.: On-probe pyrolysis desorption electrospray ionization (DESI) mass spectrometry for the analysis of non-volatile pyrolysis products. J. Anal. Appl. Pyrolysi. 80, 353–359 (2007) 10. Chen, H., Talaty, N.N., Takats, Z., Cooks, R.G.: Desorption electrospray ionization mass spectrometry for high-throughput analysis of pharmaceutical samples in the ambient environment. Anal. Chem. 77, 6915–6927 (2005) 11. Wiseman, J.M., Ifa, D.R., Song, Q., Cooks, R.G.: Tissue imaging at atmospheric pressure using desorption electrospray ionization (DESI) mass spectrometry. Angew. Chem. Int. Ed. 45, 7188–7192 (2006) 12. Laskin, J., Laskin, A., Roach, P.J., Slysz, G.W., Anderson, G.A., Nizkorodov, S.A., Bones, D.L., Nguyen, L.Q.: High-resolution desorption electrospray ionization mass spectrometry for chemical characterization of organic aerosols. Anal. Chem. 82, 2048–2058 (2010) 13. Kaur-Atwal, G., Weston, D.J., Green, P.S., Crosland, S., Bonner, P.L.R., Creaser, C.S.: Analysis of tryptic peptides using desorption electrospray ionisation combined with ion mobility spectrometry/mass spectrometry. Rapid Commun. Mass Spectrom. 21, 1131–1138 (2007) 14. Denes, J., Katona, M., Hosszu, A., Czuczy, N., Takats, Z.: Analysis of biological fluids by direct combination of solid phase extraction and desorption electrospray ionization mass spectrometry. Anal. Chem. 81, 1669–1675 (2009)
292
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
15. Bereman, M.S., Nyadong, L., Fernandez, F.M., Muddiman, D.C.: Direct high-resolution peptide and protein analysis by desorption electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Rapid Commun. Mass Spectrom. 20, 3409–3411 (2006) 16. Miao, Z., Chen, H.: Direct analysis of liquid samples by desorption electrospray ionization-mass spectrometry (DESI-MS). J. Am. Soc. Mass Spectrom. 20, 10–19 (2009) 17. Moore, B.N., Hamdy, O., Julian, R.R.: Protein structure evolution in liquid DESI as revealed by selective noncovalent adduct protein probing. Int. J. Mass Spectrom. 330/332, 220–225 (2012) 18. Miao, Z., Wu, S., Chen, H.: The study of protein conformation in solution via direct sampling by desorption electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 21, 1730–1736 (2010) 19. Ma, X., Zhao, M., Lin, Z., Zhang, S., Yang, C., Zhang, X.: Versatile platform employing desorption electrospray ionization mass spectrometry for high-throughput analysis. Anal. Chem. 80, 6131–6136 (2008) 20. Chipuk, J.E., Brodbelt, J.S.: Transmission mode desorption electrospray ionization. J. Am. Soc. Mass Spectrom. 19, 1612–1620 (2008) 21. Zhang, Y., Chen, H.: Detection of saccharides by reactive desorption electrospray ionization (DESI) using modified phenylboronic acids. Int. J. Mass Spectrom. 289, 98–107 (2010) 22. Zhang, Y., Yuan, Z., Dewald, H.D., Chen, H.: Coupling of liquid chromatography with mass spectrometry by desorption electrospray ionization (DESI). Chem. Commun. 47, 4171–4173 (2011) 23. Sun, X., Miao, Z., Yuan, Z., Harrington, P.B., Colla, J., Chen, H.: Coupling of single droplet micro-extraction with desorption electrospray ionization-mass spectrometry. Int. J. Mass Spectrom. 301, 102–108 (2011) 24. Ferguson, C.N., Benchaar, S.A., Miao, Z., Loo, J.A., Chen, H.: Direct ionization of large proteins and protein complexes by desorption electrospray ionization-mass spectrometry. Anal. Chem. 83, 6468– 6473 (2011) 25. Miao, Z., Chen, H., Liu, P., Liu, Y.: Development of submillisecond time-resolved mass spectrometry using desorption electrospray ionization. Anal. Chem. 83, 3994–3997 (2011) 26. Li, J., Dewald, H.D., Chen, H.: Online coupling of electrochemical reactions with liquid sample desorption electrospray ionization-mass spectrometry. Anal. Chem. 81, 9716–9722 (2009) 27. Pan, N., Liu, P., Cui, W., Tang, B., Shi, J., Chen, H.: Highly efficient ionization of phosphopeptides at low pH by desorption electrospray ionization mass spectrometry. Analyst 138, 1321–1324 (2013) 28. Zhang, Y., Cui, W., Zhang, H., Dewald, H.D., Chen, H.: Electrochemistryassisted top-down characterization of disulfide-containing proteins. Anal. Chem. 84, 3838–3842 (2012)
29. Zhang, Y., Dewald, H.D., Chen, H.: Online mass spectrometric analysis of proteins/peptides following electrolytic cleavage of disulfide bonds. J. Proteome Res. 10, 1293–1304 (2011) 30. Perry, R.H., Splendore, M., Chien, A., Davis, N.K., Zare, R.N.: Detecting reaction intermediates in liquids on the millisecond time scale using desorption electrospray ionization. Angew. Chem. Int. Ed. 50, 250–254 (2011) 31. Chen, H., Cotte-Rodriguez, I., Cooks, R.G.: cis-Diol functional group recognition by reactive desorption electrospray ionization (DESI). Chem. Commun. 6, 597–599 (2006) 32. Kuhlmann, F.E., Apffel, A., Fischer, S.M., Goldberg, G., Goodley, P.C.: J. Am. Soc. Mass Spectrom. 6, 1221–1225 (1995) 33. Takats, Z., Wiseman, J.M., Gologan, B., Cooks, R.G.: Electrosonic spray ionization. A gentle technique for generating folded proteins and protein complexes in the gas phase and for studying ion–molecule reactions at atmospheric pressure. Anal. Chem. 76, 4050–4058 (2004) 34. Zubarev, R.A., Kelleher, N.L., McLafferty, F.W.: Electron capture dissociation of multiply charged protein cations. A nonergodic process. J. Am. Chem. Soc. 120 (1998) 35. Stutzman, J.R., McLuckey, S.A.: Ion/ion reactions of MALDI-derived peptide ions: increased sequence coverage via covalent and electrostatic modification upon charge inversion. Anal. Chem. 84, 10679–10685 (2012) 36. Iavarone, A.T., Williams, E.R.: J. Am. Chem. Soc. 125, 2319–2327 (2003) 37. Liu, Y., Miao, Z., Lakshmanan, R., Loo, R.R.O., Loo, J.A., Chen, H.: Signal and charge enhancement for protein analysis by liquid chromatography-mass spectrometry with desorption electrospray ionization. Int. J. Mass Spectrom. 325/327, 161–166 (2012) 38. Streuli, C.A.: Titrations in Nonaqueous Solvents. Anal. Chem. 36, 363–369 (1964) 39. Sklenarova, H., Chocholous, P., Koblova, P., Zahalka, L., Satinsky, D., Matysova, L., Solich, P.: High-resolution monolithic columns—a new tool for effective and quick separation. Anal. Bional. Chem. 405, 2255–2263 40. Tanaka, N., Kobayashi, H.: Monolithic columns for liquid chromatography. Anal. Bioanal. Chem. 376, 298–301 (2003) 41. Morlock, G., Ueda, Y.: New coupling of planar chromatography with direct analysis in real time mass spectrometry. J. Chromatogr. A 1143, 243–251 (2007) 42. Eberherr, W., Buchberger, W., Hertsens, R., Klampfl, C.W.: Investigations on the coupling of high-performance liquid chromatography to direct analysis in real time mass spectrometry. Anal. Chem. 82, 5792– 5796 (2010)
J. Am. Soc. Mass Spectrom. (2014) 25:286Y292 DOI: 10.1007/s13361-013-0763-1
RESEARCH ARTICLE
A New Splitting Method for Both Analytical and Preparative LC/MS Yi Cai, Daniel Adams, Hao Chen Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA
Abstract. This paper presents a novel splitting method for liquid chromatography/ mass spectrometry (LC/MS) application, which allows fast MS detection of LCMS separated analytes and subsequent online analyte collection. In this approach, a PEEK capillary tube with a micro-orifice drilled on the tube side wall is used to connect with LC column. A small portion of LC eluent emerging from the orifice can be directly ionized by desorption electrospray ionization (DESI) with LC negligible time delay (6~10 ms) while the remaining analytes exiting the tube outlet can be collected. The DESI-MS analysis of eluted compounds shows eluent narrow peaks and high sensitivity because of the extremely small dead volume of the orifice used for LC eluent splitting (as low as 4 nL) and the freedom to choose favorable DESI spray solvent. In addition, online derivatization using reactive DESI is possible for supercharging proteins and for enhancing their signals without introducing extra dead volume. Unlike UV detector used in traditional preparative LC experiments, this method is applicable to compounds without chromophores (e.g., saccharides) due to the use of MS detector. Furthermore, this splitting method well suits monolithic column-based ultra-fast LC separation at a high elution flow rate of 4 mL/min. Key words: LC/MS, Eluent splitting, DESI, Protein supercharging, Ultra-fast LC
DESI spray
Received: 23 August 2013/Revised: 18 September 2013/Accepted: 28 September 2013/Published online: 20 November 2013
Introduction
S
plitting the eluent in liquid chromatography/mass spectrometry (LC/MS) experiments is often necessary under the circumstance when the mobile phase flow rate is too high for MS ionization. For instance, electrospray ionization (ESI), a common ionization method for coupling LC with MS, requires an optimal sample infusion rate at μL/min level while the mobile phase flow rate for chromatographic separation using regular analytical LC columns is in the range of 1~2 mL/min. Higher flow rates (up to 9 mL/ min) are in need for ultra-fast LC separation using monolithic columns [1, 2]. In addition, post-column splitting is also needed when the remaining portion of LC eluent after MS detection needs to be collected for preparative purpose. Typically, postcolumn splitting of LC eluent can be made simply using a Tee splitter in which one of the split streams goes to MS for detection and the other one can go to waste or to a second Electronic supplementary material The online version of this article (doi:10.1007/s13361-013-0763-1) contains supplementary material, which is available to authorized users. Correspondence to: Hao Chen; e-mail: [email protected]
detector [3, 4]. However, the connection capillary bridging the Tee and MS would introduce dead volume and could cause peak broadening [5, 6]. The addition of a splitter could also increase the dead volume, the column back pressure and the cost of experiment. In addition, there is a time delay for MS detection because of the significantly reduced flow rate of the split eluent flowing into the MS source (the flow rate can be dropped by as much as 100 times). Therefore, it is practically difficult to collect purified analytes in the other “waste” stream (because the analyte in the “waste” stream could flow out even earlier than being detected by MS). Thus, a new splitting method with fast MS detection and reduced dead volume is desired, to assist both online detection and online sample collection. Desorption electrospray ionization (DESI) originally developed by Cooks has been introduced for direct sample ionization with little or no sample preparation [7–15]. Besides analysis of solid samples, in our and other laboratories, DESI has been extended for direct liquid sample analysis [16–29]. In this study, we present a new splitting interface for LC/MS applications based on fast DESI ionization capability, which allows MS monitoring of the LC eluent with minimal time delay (in milliseconds) and online collection of the major portion of separated analytes for preparation purpose.
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
287
alcohol (m-NBA, ≥ 99.5 %), N-acetyl-D-glucosamine ( ≥ 99 %), maltohexaose, sulfadiazine (HPLC grade), sulfamerazine (HPLC grade, ≥ 98.8 %), and sulfaquinoxaline (HPLC grade, ≥ 96 %) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid was purchased from Fisher Chemicals (Pittsburgh, PA, USA). HPLC-grade methanol was also purchased from Fisher Chemicals. HPLC-grade acetonitrile was purchased from EMD Chemicals Inc. (Billerica, MA, USA).
In this approach, a PEEK capillary tube carries a microdrilled orifice on its side wall (Scheme 1) and is connected with the LC column outlet. When the LC eluent flows through the PEEK tube, a small aliquot of eluent emerges out of the orifice and can be directly sampled and ionized by DESI while the remaining analytes exiting from the tube outlet can be collected and further characterized. Advantages of such a new splitting method in combination with DESI-MS detection are severalfold. First, it minimizes the dead volume (as low as 4 nL) involved in LC eluent splitting because there is no need to use a connection tubing to bridge the splitter and MS. Thus, the occurrence of peak broadening can be avoided and high detection sensitivity can be achieved. Second, due to the prompt DESI detection of separated analytes, it is convenient to collect purified analytes from the PEEK tube outlet. This capability expands the applications of preparative liquid chromatography. Traditionally, such a collection is facilitated by using a UV detector placed between the LC column and the collection sample reservoir. In this case, MS detection allows detecting and collecting UV-transparent compounds following LC/MS analysis. Third, reactive DESI [30, 31] can be used for online derivatization, for example, for supercharging proteins without introducing an extra dead volume. Last, the application of this proposed splitting method with DESI-MS detection to monolithic column-based separation, for which a high mobile-phase flow rate is needed for ultra-fast elution, was demonstrated to be viable.
LC Separation Conditions A commercial PerkinElmer HPLC system (Perkin Elmer, Shelton, CT, USA) was used throughout the experiments. For the protein mixture separation, a Waters XB Bridge TM 300 C4 column (4.6 mm × 150 mm) was employed with a trifluoroacetic acid (TFA)-containing mobile phase of ACN:H2O:TFA (30:70:0.1 by volume) being used. The mobile phase flow rate used was 1.0 mL/min. For the saccharide mixture separation, an Agilent (Santa Clara, CA, USA) ZORBAX ODS C18 column (4.6 mm × 250 mm) was adopted with 0.1 % FA in H2O used as the mobile phase at the flow rate of 1.0 mL/min. For the mixture of the sulfonamides, a monolithic C18 column (Phenomenex (Torrance, CA, USA), Onyx, 100 × 4.6 mm) was chosen. A gradient elution program from 100 % A down to 90 % A in 4 min was used (mobile phase composition A: water and B: ACN) with a elution flow rate of 4 mL/min. A 20 μL injection loop was used for sample loading.
Experimental Chemicals
DESI-MS Detection
Insulin (from bovine pancreas, HPLC grade), ubiquitin (from bovine erythrocytes), trifluoroacetic acid (TFA), 3-nitrobenzyl
A home-built liquid DESI ion source with a Thermo Finnigan (San Jose, CA, USA) LCQ DECA ion trap mass
N2
DESI spray probe
LC column
. ...
Sample mixture
DESI spray 530 µm
. PEEK tubing
MS . ... .. Orifice (i.d. 350 µm)
.
510 µm
Outlet
PEEK tubing
Scheme 1. Apparatus of a new LC/MS post-column splitting method in which a DESI spray probe is used to ionize analytes of interest in the eluent emerging out of a micro-drilled orifice from the PEEK tube. The remaining portion of eluent exiting the PEEK tube outlet can be collected for preparative LC. Inset illustrates the zoomed-in PEEK tube with the orifice
288
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
spectrometer (Scheme 1) was used throughout the experiments. A short piece of PEEK tube (i.d. 510 μm; wall thickness: 530 μm; length: 3 cm) with a micro-drilled orifice (i.d. 350 μm) was connected to the LC column. The orifice was located in the tube 2 cm downstream from the LC column and the tube outlet was slightly bent downward to facilitate sample collection. The sample eluent flowing out of the orifice underwent interactions with the charged microdroplets generated from DESI spray for ionization. Unless specified, the spray solvent for DESI was CH3OH/H2O/HOAc (50:50:1 by volume) and injected at 10 μL/min with 5 kV applied to the spray solvent. The DESI spray probe was placed above the orifice and the distance between the probe and orifice was about 1–2 mm. The orifice was placed approximately 1 cm away from the MS inlet.
demonstration of the feasibility of this proposed splitting method in conjunction with DESI detection for LC/MS coupling. For separation, a reversed phase C4 column with mobile phase of ACN:H2O:TFA (30:70:0.1 by volume) was employed. TFA is often used as a mobile phase modifier to promote protein separation as TFA not only can adjust pH but also serves as an ion-pairing agent. But TFA is not an “ESI-friendly” reagent because of its signal suppression effect [32]. With the eluent from LC column flowing through the PEEK tube at a flow rate of 1.0 mL/min, part of the eluent (30 % in this prototype experiment) emerging from the PEEK orifice was ionized by DESI. As shown in the extracted ion chromatograms (EICs, Figure 1a and b), two proteins were well separated and the peak widths for insulin and ubiquitin are approximately 0.7 and 1.0 min, respectively. The resulting DESI-MS spectra (Figure 1c and d) also clearly display the ionized individual proteins with multiple charge distribution. In addition, it takes only 10 ms for the sample eluent to go through the micro-drilled orifice, which is calculated based on the flow rate of eluent through
Results and Discussion In this study, a protein mixture of insulin and ubiquitin (100 μM each) was first chosen as a test sample for LC/DESI-MS
(a)
100
0 100
LC/Reactive DESI-MS, with m-NBA +4
+5
EIC of insulin NL: 1.76E6
(a’) EIC of insulin
100
NL: 2.83E7
(b) EIC of ubiquitin
0 100
+6
(b’) EIC of ubiquitin
Relative Abundance
NL: 2.69E6
0 0.0
0.5
1.0
+7
NL: 2.02E7
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0 0.0
5.5
0.5
1.0
1.5
2.0
2.5
100
+4
(c) Insulin
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Time (min)
Time (min) +5
(c’) Insulin
100
NL : 2.15E6
NL : 2.42E5
+4
50
50
0
100
600
800
1000
1200
(d) Ubiquitin
+6
+3
+5 1400
1600
1800
+3 2000
0
600
+6
+7
+5
+9
800
1000
1400
1200
1400
m/z
1600
1800
2000
0
1600
1800
+6
+10 +11 +12 +13 600
800
2000
+8
+9 50
+10 +11 +12 600
1200
+7
NL: 1.30E6
+8
50
1000
(d’) Ubiquitin 100
NL : 2.26E5
0
800
+5 1000
1200
1400
1600
1800
2000
m/z
Figure 1. EICs of (a) insulin (+4 ion) and (b) ubiquitin (+6 ion) from LC/DESI-MS analysis and the corresponding DESI-MS spectra of (c) insulin and (d) ubiquitin. EICs of (a′) insulin (+5 ion at m/z = 1148) and (b′) ubiquitin (+7 ion at m/z = 1224) from LC/ reactive DESI-MS analysis where m-NBA was doped into the DESI spray solvent and the corresponding reactive DESI-MS spectra of (c′) insulin and (d′) ubiquitin
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
the orifice (300 μL/min) and the calculated orifice dead volume of 50 nL (i.d. 350 μm; tube wall thickness: 530 μm). Therefore, DESI-MS used in this experiment virtually offers a “near-real time” monitoring of LC eluent stream in the PEEK tube. For comparison purpose, electrosonic spray ionization [33] (ESSI, a variant form of ESI that employs an ESI source with supersonic nebulization gas for better desolvation effect) was also used to detect the LC separated analytes. In the experiment, the LC eluent flow rate was reduced to 10 μL/min (an optimized flow rate for ESSI ionization) using a commercial splitter (ASI adjustable commercial splitter), for the reason that the high flow of LC eluent at 1.0 mL/min would flood the ESSI ion source. LC separation experimental conditions were kept the same as those used in LC/DESI-MS analysis mentioned above. As revealed by the resulting EIC spectra (Figure 1S-a and S-b, Supporting Information) from ESSI-MS detection, the two EIC peaks of the proteins are much wider than those recorded using LC/ DESI-MS, each of which lasts about 4 min (Figure 1S-a and S-b, Supporting Information). It is probably caused by the increased dead volume from the traditional splitting method used for ESSI ionization. Presumably because of peak broadening (or a more severe TFA ion suppression effect), the EIC intensities from ESSI-MS detection (Figure 1S-a and S-b, Supporting Information) are lower than those from DESI-MS detection (Figure 1a and b). In addition to splitting LC eluent without introducing significant dead volume to allow fast MS monitoring, our method has an additional benefit for enabling online derivatization using reactive DESI. Reactive DESI [30, 31], in which a chosen chemical reagent is doped with the DESI spray solvent, is applicable in this LC/DESI-MS method, for supercharging proteins and for enhancing their signals. Such an online supercharging experiment would not introduce an extra dead volume as the supercharging occurs during DESI ionization. The charge enhancement for proteins by supercharging is of great value for further structural analysis via top-down approaches for increasing the fragmentation efficiency [34, 35]. It is known that 3-nitrobenzyl alcohol (m-NBA) is an effective supercharging reagent [36]. Indeed, as m-NBA was doped in the DESI spray, the maximum charge state of insulin shifted from +5 to +6 and the charge with highest abundance shifted from +4 to +5 (Figure 1c′) compared with the regular DESI data (Figure 1c). Likewise, the maximum charge state of ubiquitin shifted from +12 to +13 and the charge with highest abundance shifted from +6 to +7 (Figure 1d′). Interestingly, the signals of resulting protein ions in this LC/reactive DESI-MS (Figure 1a′ and b′) are significantly enhanced in comparison to those of regular DESI (Figure 1a and b) and ESSI ionization (Figure 1S-a and S-b, Supporting Information). This phenomenon is in agreement with our previous observation [37]. Presumably, m-NBA reduces TFA dissociation [38] so that the decreased concentration of trifluoroacetate anions reduces their ion pairing interaction with protein cationic sites, a mechanism responsible for signal suppression of TFA.
289
Besides the analytical strength, another important feature of this method is to collect isolated samples following LC/ MS analysis. Indeed, with the aid of online DESI monitoring as mentioned above, the major portion of insulin and ubiquitin exiting from the PEEK tube outlet (70 %) were collected. Re-analysis of the two collected samples by ESSIMS gave rise to the spectra of isolated insulin and ubiquitin (Figure 2S-a and S-b, Supporting Information), showing no cross-talking and confirming that the two samples were completely separated and successfully collected. Conventionally, in the preparative LC experiment, the collection of LC-separated eluent is often facilitated by placing a UV detector between the LC separation column and the sample collection reservoir. However, such an approach is limited to compounds with chromophores. For those without chromophores, derivatization is in need, which is time-consuming and troublesome. By using our DESI method, this problem can be solved when MS servers as a detector in our method. As a demonstration, a saccharide mixture consisting of Nacetyl-D-glucosamine (NAG) and maltohexaose (2.5 mM each) with no or weak UV absorption was chosen as a test sample and a C18 column was used for separation. As shown in the recorded EIC spectra, NAG and maltohexaose were well separated, and each of the saccharide peak width is about 0.6 min (Figure 2a and b). Note that although there is no organic solvent used in the mobile phase (only 0.1 % FA in H2O was used as the mobile phase), the saccharides can still be well detected by DESI-MS. This is ascribed to the freedom of choosing favorable DESI spray solvent of CH3OH/H2O/HOAc (50:50:1 by volume) for sample ionization. This phenomenon agrees with previous reports [18, 24]. Following the LC/DESI-MS analysis, the remaining saccharides were online collected and re-tested with MS, which again shows the full separation of two saccharides (Figure 2c and d). Interestingly, the eluent splitting ratio provided by the PEEK tube with the orifice can be adjusted. Simply by reducing the orifice i.d. from 350 μm down to 100 μm, the splitting ratio of 3:7 can be reduced to 4:96 for which more sample can be collected after LC separation. Experiment was further conducted to evaluate the performance of the PEEK tube carrying the 100 μm i.d. orifice. In this case, the time for eluent going through the orifice channel was only 6 ms (the split flow rate: 40 μL/min; orifice dead volume: 4 nL). By still using a mixture of insulin and ubiquitin as a test sample, the EIC spectra acquired show good protein separation (Figure 3S-a and S-b, Supporting Information). In comparison to Figure 1a and b, the signals of both proteins are even higher, probably because a lesser amount of TFA is involved in the sample being ionized. Also, the proteins after LC separation were collected, and re-analysis of the collected samples show clean spectra (Figure 3S-c and S-d, Supporting Information). We quantified the yield of collection using UV spectroscopy, which shows 94.2 % ± 0.6 % average yield for insulin and 94.4 % ± 1.8 % average yield for ubiquitin (see discussion in the Supporting
290
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
[NAG + H]+
[NAG + H]+
[2NAG + H]+
(c) Collected N-acetyl-D-glucosamine
100
100
(a)LC/DESI-MS EIC of N-acetyl-D-glucosamine NL: 2.70E6 50
Relative Abundance
[NAG - H2O + H]+
[3NAG + H]+
[4NAG + H]+
0
0
200
[Maltohexaose + H]+
100
(b) LC/DESI-MS
100
300
400
500
600
700
(d) Collected maltohexaose
800
900
[Maltohexaose +
1000
H]+
EIC of maltohexaose NL: 1.75E6
50
[Maltohexaose + Na]+
223 0 0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
200
5.0
300
400
500
600
700
800
900
1000
m/z
Time (min)
Figure 2. EICs of (a) NAG and (b) maltohexaose from LC/DESI-MS analysis. MS spectra of (c) the collected NAG and (d) the collected maltohexaose. The peak at m/z 223 in (d) came from solvent background
Information). These yields are fairly close to the theoretical value of 96 % based on the splitting ratio, suggestion the
feasibility and potential of this approach for sample purification.
[Sulfamerazine + H]+
100
a) LC/DESI-MS [Sulfaquinoxaline + H]+
Relative Abundance
[Sulfadiazine + H]+
0 0.0
0.5
1.0
1.5
2.0
2.5
Sulfadiazine
Sulfaquinoxaline
100
0.5
3.5
Sulfamerazine
b) LC/UV
0.0
3.0
1.0
1.5
2.0
2.5
3.0
3.5
Time (min) Figure 3. EIC spectra of a mixture of sulfadiazine, sulfamerazine, and sulfaquinoxaline (a) from LC/DESI-MS analysis and (b) from LC/UV analysis. The LC column used was Onyx C18 monolithic column and the PEEK tube with a 100 μm i.d. orifice was utilized. The first peak in UV spectrum might be from gas bubble introduced during sample loading
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
Besides the applications to LC separation using regular analytical columns, we also examined the compatibility of our splitting method to monolithic column-based ultra-fast LC separation. Monolithic column is a kind of “single-piece column” that has been developed for fast high-throughput analysis [39]. It allows performing high-throughput analysis with good separation performance close to that of ultra-high performance LC [2, 39, 40].The key factor in such an LC experiment is that an extremely high flow rate (up to 9 mL/min) can be used with monolithic columns without causing significantly high back pressure. Thus, the separation can be completed in a short period of time. In our experiment, a mixture of sulfonamides, including sulfadiazine, sulfamerazine, and sulfaquinoxaline (100 μM each), drugs commonly used for eliminating bacteria and treating urinary tract infections, was chosen as a test sample for demonstration of the application of our splitting method to the monolithic column-based ultra-fast LC separation. As shown in EIC spectrum recorded by DESI-MS (Figure 3a), the sulfadiazine, sulfamerazine, and sulfaquinoxaline were all separated and eluted within 3 min. In comparison, when a regular reversedphase C18 column with an elution flow rate of 1 mL/min is used for the separation of the same mixture, the total elution time is 11 min. (Figure 5S, Supporting Information). This result shows that the high flow rate does help fast elution and separation. We also recorded the chromatogram using an UV detector (detection wavelength: 254 nm) for comparison (Figure 3b). The DESIMS-recorded chromatogram has slightly better resolution than that recorded by UV detector. For instance, there is about 10 % overlap of the sulfadiazine and sulfamerazine peaks in Figure 3b, while the two peaks are well resolved in the DESIMS recorded chromatogram. This might be also due to the overall small dead volume involved in our splitting method. However, attempt to split the high flow eluent of 4 mL/min down to 10 μL/min for ESSI-MS detection by the commercial splitter caused column failure because of high back pressure that exceeds the pressure tolerance of the monolithic column used. In contrast, in our split method in conjunction with DESI-MS detection, there is no back pressure issue as the PEEK tube used for splitting has an opening that is not blocked or linked with another device. In addition, for showing the real world sample applications, we also used this LC/DESI-MS for analysis of αlactalbumin tryptic digest. Beside identifying 11 separated peptides in the digest, we were able to successfully isolate one peptide containing an intramolecular disulfide bond resulting from protein disulfide bond scrambling during trypsin digestion at basic pH (this peptide is of interest because it is useful for studying how pH affects disulfide bond scrambling in proteins, an important issue in proteomics. See detailed discussion in Supporting Information).
Conclusions This paper reports a new and versatile splitting method for LC/MS coupling, as an additional example of combining LC with MS using ambient ionization methods [22, 37, 41, 42].
291
Premium analytical performance of DESI and successful sample purification with high collection yield was demonstrated. Narrow peak width, high sensitivity, no back pressure issue, and low cost are among the striking features of the presented methodology. Owing to the capability of DESI for ionizing both small organic molecules and high-mass proteins, this method would have wide applications in bioanalysis.
Acknowledgments The authors acknowledge support for this work by NSF Career Award (CHE-1149367).
References 1. Su, Z.H., Zou, G.A., Preiss, A., Zhang, H.W., Zou, Z.M.: Online identification of the antioxidant constituents of traditional Chinese medicine formula Chaihu-Shu-Gan-San by LC-LTQ Orbitrap mass spectrometry and microplate spectrophotometer. J. Pharmaceut. Biomed. Anal. 53, 454–461 (2010) 2. Sangoi, M.S., Todeschini, V., Steppe, M.: Fesoterodine stress degradation behavior by liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry. Talanta 84, 1068–1079 (2011) 3. Schiavo, S., Ebbel, E., Sharma, S., Matson, W., Kristal, B.S., Hersch, S., Vouros, P.: Metabolite identification using a nanoelectrospray LC-ECarray-MS integrated system. Anal. Chem. 80, 5912–5923 (2008) 4. Andrews, C.L., Li, F., Yang, E., Yu, C.-P., Vouros, P.: Incorporation of a nanosplitter interface into an LC-MS-RD system to facilitate drug metabolism studies. J. Mass Spectrom. 41, 43–49 (2006) 5. Camenzuli, M., Goodie, T.A., Bassanese, D.N., Francis, P.S., Barnett, N.W., Ritchie, H., LaDine, J., Shalliker, R.A., Conlan, X.A.: The use of parallel segmented outlet flow columns for enhanced mass spectral sensitivity at high chromatographic flow rates. Rapid Commun. Mass Spectrom. 26, 943–949 (2012) 6. Andrews, C.L., Yu, C.-P., Yang, E., Vouros, P.: Improved liquid chromatography-Mass spectrometry performance in quantitative analysis using a nanosplitter interface. J. Chromatogr. A 1053, 151–159 (2004) 7. Harris, G.A., Galhena, A.S., Fernandez, F.M.: Ambient sampling/ ionization mass spectrometry: applications and current trends. Anal. Chem. 83, 4508–4538 (2011) 8. Douglass, K.A., Venter, A.R.: Protein analysis by desorption electrospray ionization mass spectrometry and related methods. J. Mass Spectrom. 48, 553–560 (2013) 9. Zhang, S., Shin, Y.-S., Mayer, R., Basile, F.: On-probe pyrolysis desorption electrospray ionization (DESI) mass spectrometry for the analysis of non-volatile pyrolysis products. J. Anal. Appl. Pyrolysi. 80, 353–359 (2007) 10. Chen, H., Talaty, N.N., Takats, Z., Cooks, R.G.: Desorption electrospray ionization mass spectrometry for high-throughput analysis of pharmaceutical samples in the ambient environment. Anal. Chem. 77, 6915–6927 (2005) 11. Wiseman, J.M., Ifa, D.R., Song, Q., Cooks, R.G.: Tissue imaging at atmospheric pressure using desorption electrospray ionization (DESI) mass spectrometry. Angew. Chem. Int. Ed. 45, 7188–7192 (2006) 12. Laskin, J., Laskin, A., Roach, P.J., Slysz, G.W., Anderson, G.A., Nizkorodov, S.A., Bones, D.L., Nguyen, L.Q.: High-resolution desorption electrospray ionization mass spectrometry for chemical characterization of organic aerosols. Anal. Chem. 82, 2048–2058 (2010) 13. Kaur-Atwal, G., Weston, D.J., Green, P.S., Crosland, S., Bonner, P.L.R., Creaser, C.S.: Analysis of tryptic peptides using desorption electrospray ionisation combined with ion mobility spectrometry/mass spectrometry. Rapid Commun. Mass Spectrom. 21, 1131–1138 (2007) 14. Denes, J., Katona, M., Hosszu, A., Czuczy, N., Takats, Z.: Analysis of biological fluids by direct combination of solid phase extraction and desorption electrospray ionization mass spectrometry. Anal. Chem. 81, 1669–1675 (2009)
292
Y. Cai et al.: A New Analytical and Preparative LC/MS Method
15. Bereman, M.S., Nyadong, L., Fernandez, F.M., Muddiman, D.C.: Direct high-resolution peptide and protein analysis by desorption electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Rapid Commun. Mass Spectrom. 20, 3409–3411 (2006) 16. Miao, Z., Chen, H.: Direct analysis of liquid samples by desorption electrospray ionization-mass spectrometry (DESI-MS). J. Am. Soc. Mass Spectrom. 20, 10–19 (2009) 17. Moore, B.N., Hamdy, O., Julian, R.R.: Protein structure evolution in liquid DESI as revealed by selective noncovalent adduct protein probing. Int. J. Mass Spectrom. 330/332, 220–225 (2012) 18. Miao, Z., Wu, S., Chen, H.: The study of protein conformation in solution via direct sampling by desorption electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 21, 1730–1736 (2010) 19. Ma, X., Zhao, M., Lin, Z., Zhang, S., Yang, C., Zhang, X.: Versatile platform employing desorption electrospray ionization mass spectrometry for high-throughput analysis. Anal. Chem. 80, 6131–6136 (2008) 20. Chipuk, J.E., Brodbelt, J.S.: Transmission mode desorption electrospray ionization. J. Am. Soc. Mass Spectrom. 19, 1612–1620 (2008) 21. Zhang, Y., Chen, H.: Detection of saccharides by reactive desorption electrospray ionization (DESI) using modified phenylboronic acids. Int. J. Mass Spectrom. 289, 98–107 (2010) 22. Zhang, Y., Yuan, Z., Dewald, H.D., Chen, H.: Coupling of liquid chromatography with mass spectrometry by desorption electrospray ionization (DESI). Chem. Commun. 47, 4171–4173 (2011) 23. Sun, X., Miao, Z., Yuan, Z., Harrington, P.B., Colla, J., Chen, H.: Coupling of single droplet micro-extraction with desorption electrospray ionization-mass spectrometry. Int. J. Mass Spectrom. 301, 102–108 (2011) 24. Ferguson, C.N., Benchaar, S.A., Miao, Z., Loo, J.A., Chen, H.: Direct ionization of large proteins and protein complexes by desorption electrospray ionization-mass spectrometry. Anal. Chem. 83, 6468– 6473 (2011) 25. Miao, Z., Chen, H., Liu, P., Liu, Y.: Development of submillisecond time-resolved mass spectrometry using desorption electrospray ionization. Anal. Chem. 83, 3994–3997 (2011) 26. Li, J., Dewald, H.D., Chen, H.: Online coupling of electrochemical reactions with liquid sample desorption electrospray ionization-mass spectrometry. Anal. Chem. 81, 9716–9722 (2009) 27. Pan, N., Liu, P., Cui, W., Tang, B., Shi, J., Chen, H.: Highly efficient ionization of phosphopeptides at low pH by desorption electrospray ionization mass spectrometry. Analyst 138, 1321–1324 (2013) 28. Zhang, Y., Cui, W., Zhang, H., Dewald, H.D., Chen, H.: Electrochemistryassisted top-down characterization of disulfide-containing proteins. Anal. Chem. 84, 3838–3842 (2012)
29. Zhang, Y., Dewald, H.D., Chen, H.: Online mass spectrometric analysis of proteins/peptides following electrolytic cleavage of disulfide bonds. J. Proteome Res. 10, 1293–1304 (2011) 30. Perry, R.H., Splendore, M., Chien, A., Davis, N.K., Zare, R.N.: Detecting reaction intermediates in liquids on the millisecond time scale using desorption electrospray ionization. Angew. Chem. Int. Ed. 50, 250–254 (2011) 31. Chen, H., Cotte-Rodriguez, I., Cooks, R.G.: cis-Diol functional group recognition by reactive desorption electrospray ionization (DESI). Chem. Commun. 6, 597–599 (2006) 32. Kuhlmann, F.E., Apffel, A., Fischer, S.M., Goldberg, G., Goodley, P.C.: J. Am. Soc. Mass Spectrom. 6, 1221–1225 (1995) 33. Takats, Z., Wiseman, J.M., Gologan, B., Cooks, R.G.: Electrosonic spray ionization. A gentle technique for generating folded proteins and protein complexes in the gas phase and for studying ion–molecule reactions at atmospheric pressure. Anal. Chem. 76, 4050–4058 (2004) 34. Zubarev, R.A., Kelleher, N.L., McLafferty, F.W.: Electron capture dissociation of multiply charged protein cations. A nonergodic process. J. Am. Chem. Soc. 120 (1998) 35. Stutzman, J.R., McLuckey, S.A.: Ion/ion reactions of MALDI-derived peptide ions: increased sequence coverage via covalent and electrostatic modification upon charge inversion. Anal. Chem. 84, 10679–10685 (2012) 36. Iavarone, A.T., Williams, E.R.: J. Am. Chem. Soc. 125, 2319–2327 (2003) 37. Liu, Y., Miao, Z., Lakshmanan, R., Loo, R.R.O., Loo, J.A., Chen, H.: Signal and charge enhancement for protein analysis by liquid chromatography-mass spectrometry with desorption electrospray ionization. Int. J. Mass Spectrom. 325/327, 161–166 (2012) 38. Streuli, C.A.: Titrations in Nonaqueous Solvents. Anal. Chem. 36, 363–369 (1964) 39. Sklenarova, H., Chocholous, P., Koblova, P., Zahalka, L., Satinsky, D., Matysova, L., Solich, P.: High-resolution monolithic columns—a new tool for effective and quick separation. Anal. Bional. Chem. 405, 2255–2263 40. Tanaka, N., Kobayashi, H.: Monolithic columns for liquid chromatography. Anal. Bioanal. Chem. 376, 298–301 (2003) 41. Morlock, G., Ueda, Y.: New coupling of planar chromatography with direct analysis in real time mass spectrometry. J. Chromatogr. A 1143, 243–251 (2007) 42. Eberherr, W., Buchberger, W., Hertsens, R., Klampfl, C.W.: Investigations on the coupling of high-performance liquid chromatography to direct analysis in real time mass spectrometry. Anal. Chem. 82, 5792– 5796 (2010)
