Supplemental Material
In Situ Characterization of Proteins Using Laserspray Ionization on a High-Performance
MALDI-LTQ-Orbitrap Mass Spectrometer
Bingming Chen1, Christopher B. Lietz2, Lingjun Li*1,2
1
School of Pharmacy, 2Department of Chemistry, University of Wisconsin-Madison, 777
Highland Avenue, Madison, Wisconsin, 53705, United States
J. Am. Soc. Mass Spectrom.
Table of Content
1. Experimental: reagents and sample preparation
2. Supplementary figure legends
3. Supplementary figures
a. Supplementary Figure 1
b. Supplementary Figure 2
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Experimental: reagents and sample preparation
All reagents and standards were used without additional purification. Acetonitrile (ACN),
methanol and formic acid were purchased from Fisher Scientific (Pittsburgh, PA, USA). 2nitrophloroglucinol (2-NPG) was purchased from Sigma Aldrich Inc. (St. Louis, MO, USA).
Bovine insulin and lysozyme standards were purchased from Promega (Madison, WI, USA), and
peptide standard (bradykinin) was purchased from American Peptide Company (Sunnyvale, CA,
USA).
For analysis of standards, 0.5µL bradykinin (10µg/mL, dissolved in H2O, 0.1% FA),
substance P (10µg/mL, dissolved in H2O, 0.1% FA), insulin (10µg/mL, 10µg/mL, dissolved in
1:1 ACN:H2O, 0.1% FA) or lysozyme (214µg/mL, 10µg/mL, dissolved in 1:1 ACN:H2O, 0.1%
FA) standards was mixed with 0.5µL of 12.5µg/mL 2-NPG matrix (dissolved in 7:3 ACN:H2O,
0.025%FA) on MALDI target plate. For substance P, 20mg/mL of α-cyano-4-hydroxycinnamic
acid (dissolved in 1:1 ACN:H2O, 0.1%FA), 150mg/mL 2,5-dihydroxybenzoic acid (dissolved in
1:1 MeOH: H2O, 0.1%FA) and 20mg/mL sinapinic acid (dissolved in 1:1 ACN:H2O, 0.1%FA)
were also used as matrices to compare with 2-NPG. For tissue analysis, animal experiments were
conducted following institutional guidelines (UW-Madison IACUC). Brains from female
Sprague-Dawley rats were dissected, embedded in gelatin (100mg/mL in MilliQ water), snap
frozen in dry ice, and stored at -80 ºC. The frozen brain was cryosectioned into 12 µm slices and
thaw mounted onto microscope slides (75x25x1mm). The slides with tissues were washed in 70%
ethanol for 1 minute to remove lipid and dehydrated at room temperature for 30 minutes. 2-NPG
matrix was spotted onto tissue surface by pipet for profiling purposes. For imaging, matrix was
applied by airbrush at a density of 5µL/mm2: the airbrush was held at 20cm from the slide. Five
coats of matrices were applied at medium flow rate with 30 sec drying time in between.
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Supplementary Figure Legends
Supplementary Figure 1. Optimization of laserspray ionization conditions for producing
multiply charged ions in the MALDI source. (a) Highest mass and highest charge state detected
at different 2-NPG concentrations (5, 10, 12.5, 15, 20 mg/mL), (b) Percentage of multiply
charged ions produced among all peaks with S/N larger than 3 at different 2-NPG concentrations
(5, 10, 12.5, 15, 20 mg/mL). (c) Highest mass molecule and highest charge detected with
different formic acid percentages (0%, 0.025%, 0.1%, 1%), (d) Percentage of multiply charged
ions produced among all peaks with S/N larger than 3 with different formic acid percentages (0%,
0.025%, 0.1%, 1%). The best conditions are highlighted in yellow shading. Error bar represents
the standard deviation of three replicates.
Supplementary Figure 2. Comparisons of matrix effects on tissue profiling experiments. (a)
DHB tissue profiling spectrum. (b) 2-NPG tissue profiling spectrum with m/z of 1000-3000. (c)
2-NPG tissue profiling spectrum with m/z 2400-4000. Multiply charged ions are highlighted in
red.
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Chen et al., Supplementary Figure 1.
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Chen et al., Supplementary Figure 2.
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