Ruibing Chen, Mingming Ma, Limei Hui, Jiang Zhang and Lingjun Li*

Supporting Material

Measurement of Neuropeptides in Crustacean Hemolymph via MALDI Mass Spectrometry” (JASMS 2008-3862)

Ruibing Chen, Mingming Ma, Limei Hui, Jiang Zhang and Lingjun Li*

Department of Chemistry & School of Pharmacy, University of Wisconsin-Madison, 777 Highland Avenue, Madison, WI 53705-2222

Table of Contents:

Content / Pages
Table S1 / Assignments of CID fragmentation peaks of CabTRP 1a and m/z 1209 degraded from actin shown in Figure 5 / P2
Table S2 / Assignments of CID fragmentation peaks of I/LNFTHKFamide and its dimethyl labeled form as shown in Figure 6. / P3
Figure S1 / MALDI-TOF/TOF analysis of crude hemolymph. / P4
Figure S2 / SDS-PAGE separation of crude and processed hemolymph samples from C. borealis. / P5

Table S1. Assignments of CID fragmentation peaks of CabTRP 1a and m/z 1209 degraded from actin shown in Figure 5.

Figure 5a APSGFLGMRamide / Figure 5b LRVAPEESPVL
m/z / Product ion assignments / m/z / Product ion assignments
44.09 / A/L / 60.07 / S
70.09 / P/R / 70.08 / R/P
86.12 / L / 86.10 / L
104.07 / M / 112.09 / R
112.09 / R / 169.08 / AP
120.09 / F / 185.06 / SP
141.10 / a2 / 227.07 / PE
157.12 / y1-NH3 / 253.11 / b1-NH3
169.09 / b2 / 270.12 / b2
174.12 / y1 / 328.13 / y3
242.12 / PSG / 352.16 / b2-NH3
285.14 / a4 / 369.19 / b3
313.15 / b4 / 415.19 / y4
345.16 / y3-NH3 / 423.19 / b4-NH3
362.13 / y3 / 440.21 / b4
389.15 / PSGF / 514.12 / APEES
432.19 / a5 / 537.28 / b5
460.53 / b5 / 585.23 / VAPEES-28
475.24 / y4 / 638.35 / a6
502.28 / PSGFL / 666.31 / b6
545.33 / a6 / 752.30 / y7-H2O
573.33 / B6 / 770.32 / y7
630.34 / b7 / 795.34 / b7
679.40 / y6 / 841.36 / y8
761.40 / b8 / 865.38 / b8-NH3
846.44 / y8-NH3 / 882.40 / b8
916.39 / MH+-H2O / 1050.56 / a10
1078.47 / b10
1096.50 / b10+H2O

Table S2. Assignments of CID fragmentation peaks of I/LNFTHKFamide and its dimethyl labeled form as shown in Figure 6.

Putative peptide and standard
Figures 6a) and 6b) / Dimethyl labeled peptide and synthetic standard Figures6c) and 6d)
m/z / Product ion assignments / m/z / Product ion assignments
44.09 / I/L / 256.18 / b2
70.09 / N / 277.25* / HK- NH3
86.12 / I/L / 321.26 / y2
110.13 / H / 403.32 / b3
120.07 / F / 441.34* / y3-NH3
129.09 / K / 458.32 / y3
200.23* / a2 / 504.28 / b4
228.09 / b2 / 514.23 / FTHK-28
266.14 / HK / 542.42* / y4-NH3
293.11 / y2 / 559.36 / y4
367.20 / THK / 613.39 / a5
375.20 / b3 / 641.34 / b5
413.30 / y3-NH3 / 706.43 / y5
430.37 / y3 / 769.51 / a6
476.25 / b4 / 797.45 / b6
514.40 / y4-NH3 / 820.58* / y6
531.29 / y4 / 944.47 / MH+- NH3
585.31 / a5
595.33 / b5-H2O
613.29 / b5
678.33 / y5
724.89 / b6-NH3
741.39 / b6
792.39 / y6
888.38 / MH+- NH3

* Asterisksindicate peaks missing in MS/MS spectra from crude samples when compared to standards.

Figure S1. MALDI-TOF/TOF analysis of crude hemolymph. (a) MALDI mass spectrum of crude hemolymph without processing. (b) MALDI mass spectrum of 10-7 M peptide standards mixed with non-processed crude hemolymph, and three of the standards are detected.Several unidentified peaks are seen in the middle mass range, which are possibly from matrix and/or salt adducts of spiked standards. (c) MALDI mass spectrum of 10-7 M peptide standards prepared in pure water. Five peptide standards were used in this experiment, including S1:angiotensin II (DRVYIHPF, m/z 1046.5); S2:bradykinin (RPPGFSPFR m/z 1060.5); S3:angiotensin I (DRVYIHPFHL m/z 1296.7); S4:allatostatin I (APSGAQRLYGFGLamide m/z 1335.7); S5: substance P (RPKPQQFFGLMamide m/z 1347.7).

Figure S2. SDS-PAGE separation of crude and processed hemolymph samples from C. borealis. (1) 15 µl of crude hemolymph loaded directly; (3) 15 µl crude hemolymph loaded after heat treatment at 70ºCfor 10 min;Processed hemolymph samples: An aliquot of 75 µl hemolymph sample was processed individually by three precipitation methods as described in the Experimental section. The resulting samples were concentrated and visualized as follows: (9) method 1; (8) method 2; (7) method 3. Samples with the same volume were precipitated, supernatant ultra-filtered with MWCO tubes, separated and then visualized as follows: (6) method 1; (5) method 2; (4) method 3. (10) protein standards molecular weight ladder.

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