Construction of a Versatile High Precision Ambient Ionization Source for Direct Analysis and Imaging

Abstract

The design and construction of a high precision ambient ionization source matrix-assisted laser desorption electrospray ionization (MALDESI) is described in full detail including a complete parts list. The computer controlled high precision motion control system and high repetition rate Explorer laser are demonstrated during MALDESI-FT-ICR analysis of peptides and proteins ranging from 1–17 kDa. The high stability ionization source platform described herein demonstrates both the advantages of the new MALDESI source and versatility for application to numerous desorption and ionization techniques.

Introduction

The introduction and development of hybrid ambient ionization sources such as laser desorption atmospheric pressure ionization (LDAPI)[1, 2], fused droplet electrospray ionization/extraction electrospray (FD-ESI, EESI)[3, 4], direct analysis in real time (DART)[5], desorption electrospray ionization (DESI)[6], electrospray assisted laser desorption ionization (ELDI)[7–11], matrix-assisted laser desorption electrospray ionization (MALDESI)[12, 13] and infrared laser desorption electrospray ionization [14, 15] has advanced the capabilities of mass spectrometry. MALDESI for example is a pulsed ionization source that holds promise in areas ranging from top-down proteomics, tissue imaging, and ionization mechanism elucidation. The pulsed nature of MALDESI combined with its ability to generate multiply-charged ions are characteristics that are particularly well suited for Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap (LTQ-Orbi) mass spectrometry due to the inverse relationship of frequency to m/z and √(m/z), respectively. The consequence of this relationship is high resolving power, < 3 ppm mass measurement accuracy, and amenability to a multitude of tandem MS/MS techniques for bottom-up and top-down proteomics (e.g., CID, ETD, ECD, SORI, and IRMPD).[16–18] Many of these MS/MS techniques in FT-ICR and Orbitrap instruments would benefit from a pulsed ionization source where intact proteins and polypeptides with complex post-translational modifications could be carefully interrogated rather than the typical “continuous” ionization encountered during a LC-MS/MS analysis where peak widths (i.e. analysis times) are on the order of 10–30 seconds.

The advancement and acceptance of MALDESI and related hybrid ionization techniques are critically dependent on the widespread dissemination of detailed source designs such that results can be reproduced and improved in a variety of laboratories. Furthermore, there are certainly unanticipated potential applications that could benefit from novel or improved source designs. Herein we provide a detailed description and characterization of the third version of the MALDESI source utilized in this laboratory. The further improvement to this design both within our lab as well as other laboratories should enable MALDESI and related hybrid ambient ionization sources to mature to a level of analytical robustness now widely enjoyed for ESI and MALDI.

Experimental

Materials

Bradykinin, angiotensin I, melittin, glucagon, bovine ubiquitin, lysozyme, myoglobin and 2,5-dihydroxybenzoic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. HPLC-grade acetonitrile and high purity water were purchased from Burdick and Jackson (Muskegon, MI, USA). The electrospray solution was prepared by mixing acetonitrile and water 1:1 (v:v). The organic matrix solution was prepared by dissolving 150 mg DHB into 1 mL of the electrospray solution. All samples were prepared from 200 µM stock solutions and mixed 1:1 (v:v) with the matrix solution. Sample spots of 0.8 µL of the analyte matrix solution were deposited for each sample, equivalent to ~80 picomoles analyte per spot.

Profilometery Measurements

A Tencor Alpha Step profilometer (5 nm resolution) was used to measure the diameter of the laser ablation crater in a gold coated QCM electrode. The distance was measured across the width of the laser ablation craters with 3 repeats at a scan speed of 50 µm/second. The QCM electrode (International Crystal Manufacturing, Oklahoma City, OK, USA) consisted of 10 MHz A/T cut quartz crystal with a 1000 angstrom gold layer coated on a chromium base layer.

MALDESI-LTQ-FT Mass Spectrometry

MALDESI mass spectra were obtained using a hybrid LTQ-FT Ultra mass spectrometer (Thermo Electron Inc., San Jose, CA, USA and Bremen, Germany) equipped with an actively-shielded 7 T superconducting magnet (Oxford Instruments, Concord, MA, USA). The MALDESI source (Figure 1) was placed in front of the LTQ-FT fitted with a modified extended ion transfer capillary (Part#54). Solvent was electrosprayed at 2.8 kV through a 75 µm i.d. fused silica capillary (Part#40) with a 30 µm fused silica tapered PicoTip (Part#44) using a Harvard PHD-2000 syringe pump (Part#28) at a flow rate of 400 nL/minute. The analyte was laser desorbed from each sample spot actively-dried[ 19] onto a stainless steel sample target (Part#36) located directly below and between the ESI emitter and the ion transfer capillary. The stainless steel sample target was biased at 300 volts. Each mass spectrum was single-acquisition with resolving power at 400 m/z set to 200,000_fwhm and the AGC is set to 1 × 10⁶.

Figure 1.

Photograph of the new MALDESI ionization source coupled to a LTQ-FT mass spectrometer with major parts indicated and part numbers that correspond to those listed in Table 1.

Source Design

MALDESI Source Design and Construction

A new version of the MALDESI source was constructed based in part on the previous source design.[12, 13] A photo of the new source is shown in Figure 1 with major parts indicated. A complete parts list, grouped by manufacturer, is included in Table 1, with specifications for custom fabricated parts described in Table 2. A schematic of the ionization source labeled by part number is included in the Supplemental Material (Figure S1). The manual linear XYZ sample positioning stages were replaced with computer controlled motorized sample positioning stages (Parts#2–5) and control unit (Part#6) for precise sample positioning during analysis. The high speed long travel translational stages (Parts#2, 3) allow for interrogation of samples across the full length and width of a standard MALDI target (Part#36). Custom translation programming was accomplished using the supplied software (Newport, ESP); enabling one touch program execution for whole sample spot analysis (a diagram of the ablation path is shown in the Supplemental Material (Figure S2)) as well as allowing the user to operate the source remotely. The position control can be actuated linearly without program execution for spot to spot analysis, as typically performed in MALDI. The high precision (0.035 µm resolution, 100 nm increments) of the positioning system enables accurate repeatable (± 600 nm, bi-directional) positioning amenable to applications requiring a high degree of positioning control (e.g., tissue imaging).

Table 1.

MALDESI Parts List

Label	Description	Distributor	Part #	Quantity
1	Explorer Q-Switched DPSS Laser	Newport (Irvine, CA)	EXPL-349-120-1KE	1
2	High performance low profile linear stage	Newport (Irvine, CA)	436	3
3	Motorized actuator (X and Y axes)	Newport (Irvine, CA)	LTA-HS	2
4	Vernier Micrometer (Z axis)	Newport (Irvine, CA)	SM-50	1
5	Angle bracket (90 deg)	Newport (Irvine, CA)	360-90	1
6	2 Axis motion controller/driver	Newport (Irvine, CA)	ESP300-11N1N1	1
7	19 in. rack mount brackets (ESP300)	Newport (Irvine, CA)	ESP300-R	1
8	Slotted base	Newport (Irvine, CA)	B-05A	3
9	2 in. post holder	Newport (Irvine, CA)	VPH-2	3
10	2 in. ss post	Newport (Irvine, CA)	SP-2	1
11	4 in. ss post	Newport (Irvine, CA)	SP-4	1
12	6 in. ss post	Newport (Irvine, CA)	SP-6	2
13	12 in. ss post	Newport (Irvine, CA)	SP-12	2
14	Right angle post connector	Newport (Irvine, CA)	CA-1	1
15	Adjustable angle post connector	Newport (Irvine, CA)	CA-2	1
16	1 in. lens mount	Newport (Irvine, CA)	LH-1	1
17	UV fused silica plano convex lens AR10	Newport (Irvine, CA)	SPX017 + AR.10	1
18	UV enhanced aluminum mirror	Newport (Irvine, CA)	10D20AL.2	2
19	Performance plus breadboard	Thorlabs (Newton, NJ)	PBI11111	1
20	Passive support frame (27.5"H × 36"L × 30"W)	Thorlabs (Newton, NJ)	PFP51505	1
21	Periscope assembly	Thorlabs (Newton, NJ)	RS99	1
22	4 in. stainless steel post	Thorlabs (Newton, NJ)	RS4	1
23	4 in. mounting post	Thorlabs (Newton, NJ)	P4	4
24	Swivel casters (3 in.)	Grainger	1G196	4
25	Caster brake kit	Grainger	4X698	4
26	Power strip (6 plug)	Grainger	6X953	2
27	DC power supply	Analytica of Branford	103510	1
28	PHD 2000 Syringe pump	Harvard Apparatus	70–2000	1
29	Fiber-Lite Light source	Dolan Jenner	MI-150	1
30	Dell laptop computer (Pentium M, 1.4 GHz)	Dell	N/A	1
31	Dell 19 in. monitor	Dell	N/A	1
32	CCD Camera	Hitachi	KP-M1AN	1
33	Leica optical amplifier	Vashaw Scientific	312996	1
34	Leica Monozoom 7	N/A	N/A	1
35	Happauge Win-TV USB-2	Circuit City	N/A	1
36	SS 192 well MALDI plate	Applied Biosystems	4333375	1
37	RS232 to USB converter	Tiger Direct	N/A	1
38	High Voltage wire (5kV, 2 pcs. 12 in., 24 in.)	N/A	N/A	1
39	LTQ high voltage plug	Connectronics Corp.	10334-02	1
40	24 in. fused silica capillary (75 um i.d.)	Polymicro Technologies	2000019	1
41	Stainless steel union	Valco Instrument Co.	ZUIXC	1
42	Syringe adapter	Valco Instrument Co.	VISF-2	1
43	PEEK tubing sleeve orange 0.062×0.016	Upchurch Scientific	F-230	2
44	Silica tip (30um i.d. tapered)	New Objective Inc.	FS360-75-30-N-20	1
45	Hamilton gas tight syringe 100 uL	Fisher Scientific	14-813-138	1
46	Breadboard	Fabricated in house	N/A	1
47	Camera support bracket	Fabricated in house	N/A	1
48	Teflon ESI holder	Fabricated in house	N/A	1
49	Sample target high voltage clip	Fabricated in house	N/A	1
50	19 in. rack support bracket	Fabricated in house	N/A	2
51	Sample target right angle bracket	Fabricated in house	N/A	1
52	Sample plate Teflon insulator	Fabricated in house	N/A	1
53	Computer shelf	Fabricated in house	N/A	1
54	Extended capillary	Fabricated in house	N/A	1
55	Laser spacing block	Fabricated in house	N/A	1

Table 2.

Description of parts fabricated in-house

46	16 in. × 16 in. × 3/8 in. thickness aluminum (thickness may be reduced to 1/4 in.), drilled and tapped 1/4 × 20 (1" on center)
47	8 in. × 1/2 in. thickness aluminum, two piece clamp for Monozoom lens support
48	2 in. × 1/2 in. × 1/4 in. thickness Teflon; cut-out to receive a stainless steel union, threaded tube inserted across width to attach aluminum retainer and electrical contact for ESI voltage
49	1¼ in. × 1/8 in. × 1/32 in. thickness stainless steel with custom bend for electrical contact with sample target
50	16 in. × 2½ in. (1½ in. along component attachment region) × 1/4 in. thickness aluminum, slots at each end for attachment to support frame (Part#20)
51	3 in. length × 2 ½ in. height, 1/4 in. thickness aluminum, (2) vertical slots cut in short side for attachment to XYZ stage, (2) clear holes (#8) in long side for Teflon target insulator attachment
52	2½ in. × 2½ in. × 1/4 in. thickness Teflon with (2) small stainless steel posts to accommodate the sample target and (2) holes drilled and tapped 8/32 in. from bottom to attach to sample target bracket (Part#51)
53	1/4 in. thickness aluminum, cut to fit across upper cross members of support frame (Part#20)
54	2 in. extended stainless steel capillary with lug to fit LTQ atmospheric pressure interface
55	7½ in. × 3¾ in. × 1½ in. aluminum block including ½ in. slots on each end for ¼ × 20 screw attachment to the breadboard (Part#19)

The manually actuated pulsed nitrogen laser (337 nm) was replaced by the Explorer laser system (Part#1), a Q-switched diode pumped solid-state ultraviolet laser (349 nm) with first pulse suppression, precise internally measured laser power (0–120 µJ) and repetition rate control (0–5000 Hz) using the supplied software (Spectra-Physics, L-Win). The laser power, repetition rate and translation stage velocity control (0–5 mm/s) are invaluable for applications to various samples, surfaces and desorption conditions; preventing damage to solid substrate and depletion of analyte during analysis. The high resolution CCD camera (Parts#32–34) was connected using the WinTV PCI tuner (Part#35) to the laptop computer and monitor (Parts#30, 31) enabling real time on screen visualization and recording of the sample spot and tracking of the laser spot on target during laser ablation, which is particularly useful for analysis of small sample spots as well as tissue sections for imaging.

The XYZ stage (Parts#2–5, 38, 49, 51, 52), second stage laser beam positioning mirror (Part#18, mounted in the upper portion of the periscope assembly Parts#21, 11, 8), focusing lens assembly (Parts#8, 9, 11, 13, 14, 16, 17), ESI emitter assembly (Parts#8, 9, 12, 12, 15, 40–44, 48) and CCD camera assembly (Parts#8, 9, 10, 13, 32–34, 47) were all mounted onto the custom fabricated breadboard (Part#46). A schematic of the desorption and ionization region with a description of each sub-assembly are shown in Figure 2 and Figure 3. A clear hole drilled in the breadboard (Part#46) allows the passage of the laser beam from the first stage directional mirror (Parts#18, 21, 22) mounted on the main breadboard work surface (Part#19) to the second stage directional mirror (Parts#8, 11, 18, 21) mounted on the custom breadboard (Part#46). The breadboard (Part#46) with all mounted equipment was then mounted onto the vibrationally isolated main breadboard (Part#19) using 4 inch offset posts (Part#23). The laser (Part#1) mounted to a custom fabricated spacing block (Part#55), first stage laser beam positioning mirror (Part#18 mounted in lower portion of the periscope assembly, Part#21, attached to a 4 inch post, Part#22), syringe pump (Part#28), light source (Part#29) and monitor (Part#31) were mounted on the main breadboard (Part#19). The field of the main breadboard (Part#19) and custom breadboard (Part#46) were fabricated with tapped holes (1/4 × 20) on one inch center; a supply of appropriate fasteners is required to attach various components to the breadboards.

Figure 2.

Side view schematic of the ionization region of the MALDESI source with the numbers corresponding to the parts listed in Table 1.

Figure 3.

Top view schematic of the ionization region with a listing of the parts for each sub-assembly.

The high voltage power supply (Part#27) and motion controller (Part#6) were mounted to the support frame (Part#20) using custom fabricated rack mounting brackets (part#50). The high voltage power supply (Part#27) was attached to the sample target high voltage clip (Part#49) using 5 kV insulated wire (Part#38) for contact with the stainless steel sample target (Part#36) mounted on the Teflon insulator (Part#52). The keyboard, mouse and laptop computer (Part#30) were placed onto a custom fabricated shelf (part#53) situated on the upper cross beams of the support frame (Part#20). Power management was provided using two 6 plug power strips (Part#26) installed on the inside of the vertical supports on the rear of the support frame (Part#20). Prior to assembly, casters (Parts#24, 25) were installed to replace the existing adjustable feet on the support frame for mobility. The versatility of this ionization source platform allows for interchangeability of the source between instruments as well as installation of new components onto the existing platform.

Desorption and Ionization Source Versatility

The high stability ionization source platform may be implemented using various desorption and ionization techniques for application specific analysis. Existing ionization sources may be installed onto the platform due to the inherent versatility of the breadboard for roll-up accessibility. Analyte desorption may be induced by one of a number of regimes including laser desorption as in MALDESI, laser induced acoustic desorption (LIAD),[20] heated nitrogen gas as in ASAP[21] and impact of high velocity charged droplets as in DESI[6] followed by a number of post-desorption ionization methods including atmospheric pressure photo ionization (APPI)[22], atmospheric pressure chemical ionization (APCI)[23] and electrospray ionization (ESI)[24]. In addition to the hybrid ionization sources listed above, this platform is amenable to basic electrospray ionization[24] and atmospheric pressure matrix-assisted laser desorption ionization.[25]

Results and Discussion

Explorer Laser Beam Characterization

The laser beam spot size was measured at a laser power of 112 µJ (measured internally) using a gold coated quartz crystal microbalance electrode as the laser target. A photo of the QCM electrode following laser ablation is shown in Figure 4B, with an expanded view of a single ablation crater shown in Figure 4C. In these experiments, the thin gold top layer (1000 angstroms) was removed exposing the chromium base layer with some re-deposition along the perimeter of the ablated crater. The elliptical laser beam spot size (60 µm × 80 µm) was determined by measuring the actual diameter of the ablation craters using a Tencor Alpha Step profilometer. A representative scan plot is shown in Figure 4A.

Figure 4.

A) Profilometer scan plot across the width (60 µm) of the laser ablation crater in the gold QCM electrode. B) Photograph of the QCM electrode after laser desorption. C) Expanded view of an elliptical ablation crater, the diameters (60 µm × 80 µm) were measured for multiple ablation craters across the QCM electrode.

MALDESI-LTQ-FT-ICR of Peptides and Proteins

MALDESI-FT-ICR mass spectra of peptides and proteins (1 – 8.6 kDa) including bradykinin, angiotensin I, melittin, glucagon and ubiquitin each mixed with organic matrix have been shown previously.[12, 13] Comparable data was obtained using the new source; representative MALDESI-FT-ICR mass spectra of bradykinin and melittin both mixed with DHB included in Supplemental Material (Figure S3). Lysozyme and myoglobin (not shown previously) were each mixed 1:1 (v:v) with DHB, 0.8 µL was deposited onto the sample target and actively dried.[19] The ESI solution flow-rate was set to 400 nL/minute and stable electrospray was obtained. The motion controller was pre-programmed to raster the sample surface under the laser beam as illustrated in the Supplemental Material (Figure S2). The laser was actuated using computer control at a laser power of 50 µJ (measured internally) and repetition rate of 10 Hz and the motion program initiated. Multiply-charged ions were generated and detected for each peptide and protein; demonstrating a molecular weight range from 1 – 17 kDa using this source. Representative MALDESI-FT-ICR mass spectra of myoglobin and lysozyme from this experiment are shown in Figure 5, with an observed resolving power of ~ 40,000.

Figure 5.

MALDESI-FT-ICR mass spectra of 0.8 µL actively dried spot of 200 µM A) myoglobin (16.9 kDa) and B) lysozyme C (14.3 kDa) mixed 1:1 (v:v) with 150 mg/mL DHB.

The amount of material ablated during analysis was calculated using the amount of material spotted (~80 picomoles), collection time (200 ms), laser repetition rate (10 Hz, 2 shots per collection), area of the laser spot (3846 µm²) and the area of the dried spot (A = 3.14 mm²); 196 femtomoles were removed per spectrum, assuming uniform distribution of analyte and complete removal at each laser shot. The development and implementation of molecular transport devices such as the air amplifier,[26, 27] air ejector[28] and RASTIR[29] which enable the efficient transport of ions into the mass spectrometer have been investigated and may prove important to increasing sensitivity.

Liquid Drop Sample Analysis

Liquid drop analysis of 0.8 µL droplets of 200 µM ubiquitin and myoglobin mixed 1:1 (v:v) with DHB (150 mg/mL) and deposited onto the sample target, biased at 300 V, for immediate analysis is demonstrated using MALDESI with ESI post-ionization. Liquid drop laser desorption analysis with ESI post-ionization has been demonstrated previously.[11, 30] The ESI emitter was biased at 2.8 kV to electrospray 50 % acetonitrile in water at a flow rate of 400 nL/minute. The liquid sample was continuously irradiated using the Explorer laser without moving the sample target, yielding relatively constant ion abundance over the lifetime of the droplet (approximately 30 seconds). liq-MALDESI-FT-ICR mass spectra of myoglobin and ubiquitin with ESI post-ionization are shown in Supplemental Material (Figure S4), A and B, respectively.

Conclusions

The MALDESI ionization source described herein couples high stability with precision motion and laser control. This design enables high mass resolving power analysis of biological molecules including intact and top-down characterization in addition to facilitating potential imaging applications.

The mobile design provides a stable analytical platform which may be used to implement a number of desorption regimes including laser desorption (e.g., MALDESI), high velocity solvent droplets (e.g., DESI), a stream of heated nitrogen gas (e.g., ASAP) and post desorption ionization using electrospray ionization (ESI), chemical (e.g., APCI) and photon ionization (e.g., APPI) for analysis of various types and classes of molecules on multiple MS platforms. The modular configuration of the MALDESI ionization source can facilitate the substitution of parts as necessary for specific applications or budgeting constraints. Furthermore, an IR laser could be mounted to the laser table breadboard for infrared laser MALDESI applications (IR-LDESI).[14, 15]