pubs.acs.

org/acschemicalneuroscience Article

Comparative Neuropeptidomic Analysis of Food Intake via

a Multifaceted Mass Spectrometric Approach
Ruibing Chen, Limei Hui, Stephanie S. Cape, Junhua Wang, and Lingjun Li*
Department of Chemistry & School of Pharmacy, University of Wisconsin;Madison, 777 Highland Avenue, Madison, Wisconsin 53705-2222

Abstract
R
egulation of food intake and body weight is a
complex physiological process involving inter-
actions between the nervous system and peri-
pheral signals (1-3). It is suggested that three aspects
are involved in the feeding regulation, including a sensor
that monitors the level of energy, a nervous system that
receives and integrates signals from circulating hor-
mones, and an effector system that influences energy
intake and energy expenditure (4). As one of the most
important and complex classes of signaling molecules,
many neuropeptides have been demonstrated to play
critical roles in this process. For instance, several
neuropeptides present in the hypothalamus including
neuropeptide Y (NPY), orexin, galanin, proopiomela-
Feeding behavior is a fundamental aspect of energy nocortin (POMC), melanin-concentrating hormone
homeostasis and is crucial for animal survival. This (MCH), neurotensin, cholecystokinin (CCK), bombe-
process is regulated by a multitude of neurotransmitters sin, corticotropin-releasing factor, and tachykinin can
including neuropeptides within a complex neuroendo- either stimulate or decrease food intake (5-9). How-
crine system. Given the high chemical complexity and ever, the interplay among these neuropeptides and
wide distribution of neuropeptides, the precise mole- hormones at the system level is largely unknown due
cular mechanisms at the cellular and network levels to the enormous technical challenges of studying the
remain elusive. Here we report comparative neuropep- complex mammalian nervous system. In contrast, in-
tidomic analysis of brain and a major neuroendocrine vertebrate nervous systems offer simpler yet relevant
organ in a crustacean model organism in response to model systems for gaining insight into the functional
feeding. A multifaceted approach employing direct roles of neuropeptides in feeding (10-12).
tissue matrix-assisted laser desorption/ionization mass Increasing evidence suggests that many of the signal-
spectrometry (MALDI-MS), stable isotopic labeling ing molecules and pathways underlying complex beha-
of neuropeptide extracts for quantitation, and mass viors such as feeding are conserved across species and
spectrometric imaging (MSI) has been employed to animal phyla. For example, the conserved NPY signal-
obtain complementary information on the expres- ing pathway has been strongly implicated in the regula-
sion changes of a large array of neuropeptides in the tion of food intake behaviors in vertebrates and in
brain and the pericardial organ (PO) in the crab Caenorhabditis elegans (13, 14). More recently, Droso-
Cancer borealis. Multiple neuropeptides exhibited phila neuropeptide F, a human NPY homologue, was
changes in abundance after feeding, including RF- reported to mediate food signaling through a conserved
amides, Cancer borealis tachykinin-related peptides pathway (15). Furthermore, a remarkably large and
(CabTRPs), RYamides, and pyrokinins. By combining diverse group of RFamides has been characterized in
quantitative analysis of neuropeptide changes via iso- invertebrates (16, 17), and members of the RFamide
topic labeling of brain extract and MSI mapping of family have been shown to influence feeding behavior in
neuropeptides of brain slices, we identified the bound- both vertebrates and invertebrates (18). However, the
ary of the olfactory lobe (ON) and the median proto- interactions of multiple peptidergic systems at the net-
cerebrum (MPC) area as two potential feeding centers in work levels are not well understood. To study the
the crab brain. peptidergic regulation of feeding, the Cancer borealis
Keywords: Feeding, neuropeptide, Cancer borealis,
quantitation, MALDI mass spectrometric imaging Received Date: October 12, 2009
(MSI), MALDI-TOF/TOF Accepted Date: November 26, 2009
Published on Web Date: December 28, 2009

r 2009 American Chemical Society 204 DOI: 10.1021/cn900028s |ACS Chem. Neurosci. (2010), 1, 204–214
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nervous system is a particularly attractive invertebrate be combined with single-cell physiology of the feeding
preparation due to the well-defined neural circuits in the circuit to provide further insights into neuropeptide
stomatogastric nervous system (STNS) and its extensive regulation of feeding behavior at the network and
neuromodulation by various signaling molecules in- cellular levels.
cluding numerous neuropeptides (11, 19). The neuro-
peptide complement of C. borealis has been extensively Results and Discussion
studied by immunohistochemistry (20) and mass spec-
trometry (21-25). Feeding behavior is regulated by multiple neuropep-
Conventionally the identification of appetite regula- tides within the extremely complex nervous system. A
tors has been achieved by the use of peptide injections better understanding of the neural circuitry that controls
followed by measurements of food intake, molecular this process requires knowledge of the full cast of
cloning in combination with gene expression study, and molecular players and their mode of action on each of
radioimmunoassay (26-28). Recently the advancement the elements of the circuits. Here, we employed a power-
of mass spectrometry (MS)-based techniques enabled ful multifaceted MS-based platform for sensitive and
more rapid and global analysis of neuropeptide expres- high-throughput comparative analysis of a panel of
sion in response to food intake. Fricker and co-workers neuropeptides under different feeding states. Further-
reported the use of isotopic labeling with trimethyl- more, the MSI technique enables precise localization of
ammoniumbutyryl (TMAB) N-hydroxysuccinimide peptide expression and differential mapping of peptide
coupled to liquid chromatography (LC)/MS/MS for isoforms of the same peptide family. Based on this
large-scale peptidomic changes in Cpefat/fat mouse hypo- integrated methodology, a large array of neuropeptides
thalamus by food deprivation and exercise (29). can be investigated simultaneously for their roles in the
In this study, we combine direct tissue analysis feeding process.
and stable isotope labeling to investigate tissue-specific
expression and distribution of neuropeptides in crusta- Study of Peptide Expression Level Changes in
cean brain and pericardial organ (PO) upon feeding. In Brain Tissue Using Isotopic Formaldehyde La-
addition, we employ MALDI MS imaging (MSI) to beling
examine the spatial distribution of neuropeptides in the Figure 1 shows a representative MALDI-TOF/TOF
brain, which shows site-specific neuropeptide expres- spectrum obtained from a single brain extract. The
sion. Collectively, this study demonstrates that MS- identities of neuropeptides were assigned by a combina-
based large-scale quantitative peptidomic investigation tion of accurate mass matching against an in-house
enables simultaneous determination of the coordinated organ-specific neuropeptide database of C. borealis (30)
changes of a large number of neuropeptides in feeding in and collision-induced dissociation (CID) sequence-spe-
a well-defined neuronal structure. This information can cific fragmentation. A total of 51 neuropeptides that

Figure 1. Neuropeptide detection of a single brain extract on MALDI-TOF/TOF. The abundant peaks in the spectrum are labeled with their
masses and amino acid sequences. These peptides belong to several neuropeptide families including RFamide, crustacean tachykinin-related
peptide (CabTRP), RYamide, orcokinin, YRamide, SIFamide, and proctolin.

r 2009 American Chemical Society 205 DOI: 10.1021/cn900028s |ACS Chem. Neurosci. (2010), 1, 204–214
 pubs.acs.org/acschemicalneuroscience Article

Figure 2. MALDI-TOF/TOF mass spectrum of isotopic formaldehyde labeled mixture of brain extracts. Two aliquots of C18 ziptip proce-
ssed brain extract of the same volume are labeled with formaldehyde or deuterium formaldehyde in the same way and mixed in a ratio of 1:1.
The D2-formaldehyde (FD2) labeled peaks are indicated with open circles, and the H2-formaldehyde (FH2) labeled peaks are indicated with
closed circles. There are 4 Da mass differences for each incorporated label.

belong to 15 families, including RFamide, CabTRP, groups of data and their p values were included. A
A- type, B-type, and C-type allatostatins, RYamide, graphic representation of abundance ratios for each
orcokinin, orcomyotropin, proctolin, crustacean cardio- examined neuropeptide (expressed as characteristic
active peptide (CCAP), corazonin, pigment-dispersing m/z values) in fed crab versus unfed crab is provided
hormone (PDH), pyrokinin, SIFamide, and YRamide, in Figure S2, Supporting Information.
were detected in a single spectrum. Cancer borealis Tachykinin-Related Peptides
In order to accurately measure the quantitative (CabTRPs). Significant changes were observed for
changes of neuropeptide expression in animals under members from several neuropeptide families (p <
different feeding states, stable isotopic labeling with 0.01), indicated with italic type in Table 1. Two Cab-
formaldehyde was employed. This labeling technique TRPs (m/z 934.49 and m/z 964.50) were detected to be
has been reported as a fast and simple reaction that can consistently elevated in fed crab brain from 1.4- to
be applied for differential proteomic and peptidomic 4.4-fold in all five groups of preparations with mean
analyses (31). MALDI-TOF/TOF analysis of a mixture ratios of 2.1 and 2.7 (p < 0.001). The tachykinin
of differentially labeled control brain extract combined peptide family exhibits various physiological effects
in a 1:1 volume ratio showed approximately 1:1 ratios of on both the central nervous system (CNS) and periph-
each labeled peptide pair (Figure 2), thus validating the eral tissues of organisms throughout the animal king-
labeling methodology. As a result, 25 neuropeptides dom ranging from invertebrates to mammals (32-34).
from 9 families were examined in the feeding study. It was reported previously that two mammalian tachy-
Reverse labeling experiments of two pericardial organ kinin peptides, neuropeptide K and substance P, can
extract samples have also been performed, which acutely and consistently suppress feeding behavior in
showed that the H2-formaldehyde and D2-formalde- rats (35, 36). In another feeding study with goldfish, the
hyde have the same labeling efficiency (Figure S1, level of r-preprotachykinin mRNA was increased in
Supporting Information). Using this method, we exam- the brain after food intake (37). In crustaceans, tachy-
ined the quantitative changes of neuropeptide expres- kinin-related peptides have been isolated and charac-
sion in the brains from five groups of fasting and terized in both the CNS and the STNS as well as in
satiated crabs. Figure 3 shows a representative MAL- midgut endocrine cells (38-41). Previous studies
DI-TOF/TOF mass spectrum obtained from one group showed excitatory effects of both CabTRP 1a (38)
of brain extracts. Several neuropeptide families exhib- and CabTRP II (41) peptides on the pyloric motor
ited an increase in ion abundance after feeding, includ- pattern, suggesting its involvement in food processing.
ing CabTRPs, RFamides, YRamide, and a few others. The presence and distribution of both CabTRP 1a and
Table 1 lists the five groups of average abundance ratios CabTRP II in neural tissue, as well as midgut tissues,
for 25 neuropeptides present in fed crab brain versus have been documented in numerous Cancer crabs (41)
unfed crab brain. The mean ratios averaged for five and lobster Homarus americanus (40), further supporting

r 2009 American Chemical Society 206 DOI: 10.1021/cn900028s |ACS Chem. Neurosci. (2010), 1, 204–214
 pubs.acs.org/acschemicalneuroscience Article

Figure 3. Representative MALDI-TOF/TOF mass spectrum of isotopic formaldehyde labeled mixture of brain extracts from fed and unfed
crabs. Three crab brains were used to make each extract. Sample from unfed crabs was labeled with FH2 and sample from fed crabs was labeled
with FD2. The heavy labeled peaks are indicated with open circles, and the light labeled peaks are indicated with closed circles. The peak pairs
from several abundant neuropeptides are indicated and labeled with their corresponding amino acid sequences.

its potential roles associated with feeding. Here, we represent a large neuropeptide family identified in the
provide the first direct evidence that the levels of nervous systems of animals across all major phyla with
these two crustacean tachykinin-related peptides are diverse functions (46-48). The anorexigenic role of
elevated in crab brain after food intake, which suggests RFamide in feeding regulation in mice was first sug-
that this peptide family is involved with food intake in gested 20 years ago (49), followed by increasing evi-
decapod crustaceans. This observation also agrees well dence of the involvement of this peptide family in
with previous reports on the roles of tachykinin-related feeding from many other animal models (47). The
peptides (TKRPs) in feeding in various insects (42). quantitative studies described here show increased
For instance, dramatic TKRP expression level changes levels of several RFamide isoforms in crab brain after
were observed in the brain during foraging in honey bee a meal, suggesting that RFamides may also function as
probed by quantitative mass spectrometry (43). In anorexigenic signaling molecules in crab.
addition, in cockroach, starvation led to a decrease of RYamides and YRamide. RYamides FVGGSRY
the level of LemTRP-1 in the midgut, whereas its level amide (m/z 784.41) and SGFYANRYamide (m/z
in hemolymph was elevated, suggesting that LemTRP- 976.46) and YRamide HIGSLYRamide (m/z 844.48)
1 was released from midgut to hemolymph to sustain also exhibited higher abundances in fed crab. The
food consumption (44). In our previous study, we also RYamide peptide family was first discovered in the
observed higher levels of CabTRPs in circulating fluid pericardial organ of C. borealis (24) and thus far the
hemolymph in unfed crabs, suggesting that these pep- physiological effects of this family are largely un-
tides might possibly be released from midgut endocrine known. HIGSLYRamide was identified recently from
cells under starvation (45). Interestingly, we demon- the pericardial organ and sinus gland of C. productus
strate here that CabTRPs are accumulated to a higher (50), and subsequently this peptide was also found in
level in the brain after feeding compared with levels in the sinus gland, brain, and STNS of C. borealis (22, 25).
unfed crabs, which may be due to less CabTRPs being So far no physiological study has been reported for this
released into the hemolymph after feeding. peptide. The quantitative analysis of neuropeptidomes
FMRFamide Related Peptides. The abundances of in response to feeding described here showed that both
several RFamides, including NRNFLRFamide (m/z peptides exhibited an increase in relative abundance in
965.54), GAHKNYLRFamide (m/z 1104.61), SMPS- crab brain after food intake, suggesting their potential
LRLRFamide (m/z 1105.63), APQRNFLRFamide relationship with feeding.
(m/z 1147.65), GNRNFLRFamide (m/z 1022.57), Pyrokinin. Conversely, a pyrokinin (m/z 1037.55)
and AYNRSFLRFamide (m/z 1172.63), were signi- was detected to be significantly reduced in the brain
ficantly increased in the brains of fed animals com- extract from fed animals compared with the brain
pared with unfed animals, but by a lesser extent (∼1.5 extract sampled from unfed animals (p < 0.001).
fold) than the CabTRPs. RFamide-related peptides Among the nine neuropeptide families that were

r 2009 American Chemical Society 207 DOI: 10.1021/cn900028s |ACS Chem. Neurosci. (2010), 1, 204–214
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Table 1. Ratios of Neuropeptide Abundances in the Brain Tissues Collected from Fed Crabs versus Unfed Crabs (N = 5)a
m/z sequence 1 2 3 4 5 Raverage SDaverage p value

RFamide Family
965.54 NRNFLRFamide 1.35 1.12 1.14 1.49 1.65 1.35 0.22 <0.001
1022.57 GNRNFLRFamide 1.21 1.16 1.10 1.50 1.82 1.36 0.30 0.002
1104.61 GAHKNYLRFamide 1.80 1.14 1.30 1.96 2.58 1.76 0.57 <0.001
1105.63 SMPSLRLRFamide 1.18 1.32 0.91 1.44 1.33 1.23 0.20 0.009
1147.65 APQRNFLRFamide 1.25 1.07 1.04 1.48 1.84 1.34 0.33 0.007
1172.63 AYNRSFLRFamide 1.59 1.53 1.3 1.61 1.46 1.5 0.12 <0.001
1181.62 SENRNFLRFamide 0.88 1.06 0.99 1.46 1.87 1.257 0.40 0.102
1288.68 QDLDHVFLRFamide 0.75 0.67 0.87 1.01 1.05 0.87 0.16 0.033
1342.81 DVRTPALRLRFamide 1.11 1.03 0.65 1.40 1.13 1.07 0.27 0.756
Orcokinin Family
1198.55 NFDEIDRSGFamide 1.73 0.84 1.12 1.56 1.6 1.37 0.37 0.022
1270.57 NFDEIDRSGFA 0.79 1.94 0.92 2.57 1.19 1.48 0.75 0.118
1474.63 NFDEIDRSGFGFA 1.19 0.84 1.04 1.06 1.14 1.05 0.13 0.304
1502.70 NFDEIDRSGFGFV 1.19 0.88 1.17 1.02 1.13 1.08 0.12 0.124
1532.70 NFDEIDRSSFGFV 1.25 0.71 1.42 0.92 1.03 1.07 0.27 0.702
1547.68 NFDEIDRSSFGFN 1.15 0.56 1.02 0.97 1.14 0.97 0.24 0.542
1554.70 NFDEIDRTGFGFH 1.87 0.94 0.87 1.95 2.13 1.55 0.59 0.028
RYamide Family
b
784.41 FVGGSRYamide 1.87 1.05 1.30 1.80 1.50 0.39 0.009
976.46 SGFYANRYamide 1.34 1.26 1.06 1.6 1.83 1.42 0.30 0.001
CabTRPs Family
934.49 APSGFLGMRamide 2.33 1.43 1.61 1.94 3.14 2.09 0.67 <0.001
964.50 TPSGFLGMRamide n.a 1.84 1.59 3.08 4.37 2.72 1.27 <0.001
Proctolin Family
b b
649.37 RYLPT 3.80 1.31 1.47 2.19 1.39 0.038
CCAP Family
b
956.38 PFcNAFTGcamide 1.00 1.44 1.26 1.08 1.19 0.19 0.037
YRamide Family
844.48 HIGSLYRamide 8.29 1.07 2.82 1.61 3.21 3.4 2.86 0.002
Pyrokinin Family
b
1037.55 SGGFAFSPRLamide 0.86 0.58 0.70 0.88 0.75 0.14 <0.001
SIFamide Family
1381.74 GYRKPPFNGSIFamide 1.06 1.09 0.93 1.58 1.39 1.20 0.26 0.038
a b
Peptides shown in italics exhibited significant changes upon feeding (p<0.01). Not applicable.

examined in this study, pyrokinin was the only one families present in the crab brain, and the majority of
showing significant reduction in relative ion abun- members in this family did not exhibit significant changes in
dance ratio in crab brain after food intake. An electro- abundance upon feeding. Most orcokinin peptides detected
physiological study has shown that pyrokinins excite in fed and unfed animals were around 1.0 in all five groups
gastric mill circuit neurons and elicit the gastric mill of preparations. In addition, several other neuropeptides,
rhythm (chewing) in the isolated STG from C. borealis such as SIFamide and CCAP, did not change their relative
(51). Therefore, it is not surprising to see the change in abundances significantly upon feeding.
pyrokinin content in the brain after food intake. The MS-based quantitation study of multiple fa-
Further physiological experiments are needed to estab- milies of neuropeptides in crab brains offers an initial
lish the precise roles of this peptide in feeding. glimpse of the diverse assortment of neuropeptides
Peptides Not Changed in the Brain after Feeding. involved in feeding behavior and provides the basis
Orcokinin was one of the most abundant neuropeptide for subsequent biochemical and physiological studies.

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The differential expression of neuropeptide isoforms the physiological consequences of the neuropeptides
within various peptide families upon feeding also examined in this study.
provides important biological insights into possible
Spatial Mapping of Neuropeptides Involved in
functional consequences of the large peptide diversity
Feeding in C. borealis Brain by MALDI Mass
and multiplicity in the context of food intake. Some of Spectrometric Imaging
these neuropeptides may play roles in control or termi- To investigate correlation of neuropeptide expression
nation of food intake, and some of them may be and localization, MALDI MS imaging was employed to
downstream changes or indirectly affected by the map the distribution of several neuropeptides of interest
feeding states of the animals. Future studies will be in the brain. Tissues were collected from the middle
performed to test the actions of certain target neuro- region of the brain and mass spectral images of numer-
peptides on the neural circuits. In addition, behavioral ous neuropeptides were obtained. The structure and
studies followed by infusion of particular neuropep- morphology of C. borealis brain has been well studied
tides into the animal will allow us to further understand (52). As shown in Figure 4a, the main body of the

Figure 4. Neuropeptide localization in the C. borealis brain. (a) Representation of the ventral surface of the isolated brain with labeled neuropil
regions and neuronal clusters. The main body of the decapod crustacean brain consists of three contiguous brain regions, the median proto-
cerebrum (MPC), deutocerebrum (DC), and tritocerebrum (TC). The coc projects from the tritocerebrum to the thoracic ganglion (TG). Five
major neuropils in crab brain include anterior (AMPN) and posterior medial protocerebral neuropil (PMPN), olfactory lobe (ON), lateral
antenna I neuropil (LAN), and antenna II neuropil (AnN). Neuronal clusters 6 in the MPC and 9 and 10 near the ON are projected to the STNS
through the coc. (b) Optical micrograph of a brain slice on a MALDI plate coated with thin layer of DHB matrix. The coc nerve is arranged on
the top as indicated. (c) MALDI-MS images of several neuropeptides of interest from three families, including three RFamides, CabTRP 1a,
and orcokinins with DVRTPALRLRFa and NFDEIDRTGFGFH showing distinct localization from the general distribution trend of their
respective families. Note that the orientation of the panels is the same.

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decapod crustacean brain consists of three contiguous the AnN region (Figure 4c, iii), which was distinctly
brain regions, the median protocerebrum (MPC), deu- different from the other RFamides involved in feeding.
tocerebrum (DC), and tritocerebrum (TC). There are Likewise, such differential localization of neuropeptides
five major neuropils in a crab brain, anterior (AMPN) was observed for the orcokinin family as well. The
and posterior medial protocerebral neuropil (PMPN), relative ion abundance ratios of most orcokinins be-
olfactory lobe (ON), lateral antenna I neuropil (LAN), tween brain tissues from unfed and fed crabs were
and antenna II neuropil (AnN). Three of the neuronal around 1:1, and these peptides were more concentrated
clusters were indicated, 9 and 10 on the boundary of ON in AnN and were not detected in the MPC region.
and 6 between AMPN and PMPN. Figure 4b shows the However, NFDEIDRTGFGFH (m/z 1554.70) was ex-
optical micrograph of a brain slice on the MALDI plate pressed at a higher level after feeding (∼1.5-fold), and
with a thin layer of DHB matrix coating. Figure 4c interestingly this peptide was only detected in the MPC
shows several example images of neuropeptides present region and not seen in the AnN. These results demon-
in the brain, including three RFamides, two orcokinins, strate that members from the same family exhibit dis-
and CabTRP 1a. Most RFamides, such as NRNFL- tinct distribution patterns in the crustacean brain and
RFamide (m/z 965.54) and GAHKNYLRFamide (m/z that feeding induced neuropeptide expression level
1104.65), are concentrated in AMPN and PMPN changes are highly related to their localization in the
(Figure 4c, i and ii). CabTRP 1a (APSGFLGMR- brain. A single neuropeptide family may exert multiple
amide, m/z 934.49) is the most abundant peptide in the physiological functions by expressing different isoforms
crab brain, and it is extensively present in most areas of in different brain regions. As reported by Kirby and
the brain; however it has higher concentration in the Nusbaum, C. borealis brain is connected with the STNS
inner boundary of the ON (Figure 4c, iv). Previous by projection from cell clusters 6 and 7 (near AMPN and
immunocytochemical study has also shown similar dis- PMPN) and 9 and 10 (around ON) through CoG via the
tribution of these two neuropeptide families in the brain coc fiber (52), which is consistent with the results in this
of other crustacean species (53, 54). Most isoforms of study that most neuropeptides expressed at different
the orcokinin family, such as NFDEIDRSGFGFA (m/ levels after food intake are concentrated in those two
z 1474.70), are expressed in high abundance in AnN regions. Therefore, it is very likely that the brain
(Figure 4c, v), however, unlike CabTRPs and RF- regulates the STNS motor pattern to control feeding
amides, most orcokinin members are not seen in the behavior and food processing by transporting multiple
MPC region. Most isoforms from the same family are neuropeptides that are expressed in these regions. In
localized in identical brain areas, although several exce- other words, the MPC and the ON may function as
ptions are seen. For instance, one of the orcokinins major feeding regulation centers in crustaceans. To
NFDEIDRTGFGFH (m/z 1554.70, Figure 4c, vi) exhi- further understand the mechanism of food intake, it
bits a distinct distribution pattern from the other iso- will be interesting to study the level of cell-specific
forms of the family, while displaying similar spatial neuropeptide changes within the well-characterized
patterns as the RFamides and exhibiting high abun- neural network, and immunocytochemical studies and
dance in the MPC region. Another example is DVR- single cell analysis will be carried out for such purpose.
TPALRLRFamide (m/z 1342.81), which is only detec-
ted with high abundance in AnN. Neuropeptide Changes in the Pericardial Organs
The functions of signaling molecules in a complex after Feeding
neuronal structure or an organism are often related to Pericardial organs (POs) are one of the major neuro-
their locations. Based on the current study, most of these secretory sites in crustacean nervous system. Figure 5
feeding related neuropeptides including CabTRPs and shows MALDI-TOF/TOF mass spectra of direct tissue
several RFamides were detected in the MPC region or analysis of POs from unfed (A) and fed (B) animals.
around the ON boundary (Figure 4c, i, ii, and iv). In Around 20 neuropeptides were detected directly from
contrast, most of the orcokinin family members, which the PO tissue, including RYamides, RFamides, A-type
did not show changes upon feeding, were more inten- allatostatins (AST-A) and B-type allatostatins (AST-B).
sively expressed in AnN (Figure 4c, v). Furthermore, Arginine-vasopressin (AVP, 10-6 M) was added as an
MALDI mass spectral images also showed the differ- internal standard. As shown in Figure 5, the neuropep-
ential expression of family isoforms in a region-specific tide profiles from POs in both animals were almost
manner and possible association with feeding. For identical qualitatively. However, RFamides in the POs
example, in contrast to several RFamides displaying from the fed animal were detected with much lower
differences in response to feeding, several RFamide signal intensities, and the peak intensities of several
isoforms were unchanged after feeding (Table 1), such RYamides were slightly higher in the POs from the fed
as DVRTPALRLRFamide (m/z 1342.81). MALDI animal. To more accurately measure the differences in
imaging showed that this peptide was only present in neuropeptide expression in POs between unfed and fed

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Table 2. Ratios of Neuropeptide Abundances in the

PO from Fed versus Unfed Crabs (N = 11)a
m/z sequence Raverage SDratio p value

RYamide
784.41 FVGGSRYamide 0.89 0.11 0.104
976.46 SGFYANRYamide 0.80 0.16 0.014

RFamide
965.54 NRNFLRFamide 0.71 0.14 0.001
1005.57 GPRNFLRFamide 0.88 0.12 0.069
1022.57 GNRNFLRFamide 0.86 0.15 0.039
1104.61 GAHKNYLRFamide 0.57 0.07 <0.001
1146.61 GYSKNYLRFamide 0.67 0.08 0.004
1147.65 APQRNFLRFamide 0.76 0.16 0.009
1172.63 AYNRSFLRFamide 0.80 0.11 0.009
1181.62 SENRNFLRFamide 0.68 0.11 <0.001

AST-B
1293.63 STNWSSLRSAWamide 0.92 0.16 0.269
1470.70 VPNDWAHFRGSWamide 0.82 0.13 0.078
a
Peptides highlighted in italics exhibited significant changes (p <
0.01) upon feeding.

reduction among all RFamides in the POs and the

Figure 5. MALDI-TOF/TOF mass spectra of direct tissue analysis greatest increase in the brain preparations (Table 1).
comparison of the POs from (a) unfed and (b) fed crabs. Tissues
were collected from the same position of anterior region of the PO in This observation strongly suggests that GAHKNYLR-
both animals. Arginine-vasopressin (AVP) (m/z 1084.44) was added Famide may play a more important role in food intake
as an internal standard indicated with asterisk. Sixteen neuropep- compared with other RFamides. Again, the current
tides from four families were detected and labeled with their masses study demonstrates that isoforms of different amino
and amino acid sequences.
acid sequences from a single neuropeptide family may
animals, stable isotopic labeling of tissue extract was have distinct physiological functions, therefore high-
performed. Table 2 shows ratios of neuropeptide abun- lighting the unique advantages of using a mass spectro-
dances in the POs between fed and unfed animals from metry-based peptidomics approach to reveal such
11 groups of preparations. Most isoforms of RFamides isoform-specific changes in feeding.
exhibited significant reduction in abundance in fed
animals (p < 0.01). Methods
Pericardial organs can release peptide hormones to Animals and Feeding Experiments
modulate the motor patterns generated by neural Jonah crabs, Cancer borealis, were purchased from The
circuits located in the STNS. One can expect the Fresh Lobster Company (Gloucester, MA) and maintained
involvement of this important neurohemal organ without food in an artificial seawater tank at 12-13 °C for 10
with feeding by releasing certain neuropeptides into days before use. In a feeding experiment, the crabs were fed
hemolymph. Different from trends observed in brain, with small pieces of seafood until they stopped, which usually
most detected neuropeptides exhibited reduced levels took 30-45 min. Crabs were then cold anesthetized by
in the POs after food intake; presumably due to packing them on ice for 15 min. Dissection was performed
possible release of these peptides from the POs after in chilled (approximately 10 °C) physiological saline
(composition 440 mM NaCl, 11 mM KCl, 13 mM CaCl2, 26
feeding. However, due to the low concentration of
mM MgCl2, 10 mM HEPES, pH 7.4 [adjusted with NaOH]).
neuropeptides present in the circulating fluid, RFa- The details of dissection were described previously (21).
mides were rarely detected in our hemolymph pro- Mass Spectrometry and MALDI Imaging
filing experiments. Among the three families that A model 4800 MALDI-TOF/TOF analyzer (Applied Bio-
were detected in the POs, RFamides were most sig- systems, Framingham, MA) equipped with a 200 Hz, 355 nm
nificantly reduced (Table 2). Interestingly, although Nd:YAG laser was used for direct tissue analysis, brain
several different RFamides were changed upon feed- extract quantitation, and MALDI imaging. Acquisitions
ing, GAHKNYLRFamide showed the most significant were performed in positive ion reflectron mode. Instrument

r 2009 American Chemical Society 211 DOI: 10.1021/cn900028s |ACS Chem. Neurosci. (2010), 1, 204–214
 pubs.acs.org/acschemicalneuroscience Article

parameters were set using the 4000 series Explorer software Author Information
(Applied Biosystems). Mass spectra were obtained by aver-
aging 900 laser shots covering mass range m/z 500-4000. MS/ Corresponding Author
*
MS was achieved by 1 kV collision-induced dissociation To whom correspondence should be addressed. E-mail: lli@
(CID) using air. MALDI mass spectrometric imaging of C. pharmacy.wisc.edu. Phone: (608)265-8491. Fax: (608)262-
borealis brain was performed as previously described (55). 5345.
Imaging acquisition was performed using the 4800 Imaging Author Contributions
application software (www.maldi-msi.org). To generate R.C. and L.L. designed research; R.C., L.H., S.S.C., and J.
images, spectra were collected at 100 μm intervals in both W. performed research; R.C. analyzed data; R.C. and L.L.
the x and y dimensions across the surface of the sample. Each wrote the paper.
mass spectrum was generated by averaging 200 laser shots
over the mass range m/z 800-2000. Individual spectra were Funding Sources
acquired using 1.0 ns binning to yield 27 812 data points per This work was funded by National Science Foundation
spectrum. Image files were processed, and extracted ion CAREER Award (Grant CHE-0449991) and the National
images were created using the TissueView software package Institutes of Health through Grant 1R01DK071801. L.L.
(Applied Biosystems, Framingham, MA). acknowledges an Alfred P. Sloan Research Fellowship.
Sample Preparation
Direct tissue analysis was performed as described pre-
viously (21). Briefly, the tissue was rinsed in a droplet of
acidified methanol (90% methanol, 9% acetic acid, 1%
Acknowledgment
water, v/v/v), desalted in a droplet of dilute DHB solution The authors thank Dr. Amy Harms and Dr. Mike Sussman
(10 mg/mL, aqueous), and placed on the MALDI plate. A at the University of Wisconsin Biotechnology Center Mass
droplet of 0.4 μL of standard mixed DHB matrix solution Spectrometry Facility for access to the MALDI-TOF/TOF
was deposited on top of the tissue placed on the MALDI instrument. We also wish to thank Dr. Eve Marder at
target and allowed to crystallize at room temperature. Brandeis University for valuable advice and helpful discus-
Tissue extractions were performed by homogenizing in sions for preparing the paper.
cooled acidified methanol as described elsewhere (30).
The crude extracts were processed by C18 Ziptip according
to the product instructions to remove lipids and salt before Abbreviations
the formaldehyde labeling reaction. MSI, mass spectrometric imaging; STNS, stomatogastric
Quantitation Using Isotopic Labeling nervous system; PO, pericardial organ; MPC, median
A 3 μL aliquot of tissue extract from brain or PO was protocerebrum; DC, deutocerebrum; TC, tritocerebrum;
labeled in solution by adding 0.7 μL of borane pyridine AMPN, anterior medial protocerebral neuropil; PMPN,
(C5H8BN, 120 mM in 10% methanol) and then mixing with posterior medial protocerebral neuropil; ON, olfactory
formaldehyde (FH2, 15% in H2O, 0.5 μL) for samples from lobe; LAN, lateral antenna I neuropil; AnN, antenna II
unfed animals or deuterium formaldehyde (FD2, 15% in H2O, neuropil; CabTRP, Cancer borealis tachykinin-related
0.5 μL) for samples from fed animals. The samples were then peptide; AST, allatostatin; STG, stomatogastric gang-
placed in a 37 °C water bath for 20 min for the labeling lion; coc, circumoesophageal connective; CoG, com-
reaction to complete. Samples from fed and unfed animals missural ganglia; DHB, 2,5-dihydroxybenzoic acid;
were then mixed in a 1:1 ratio. Each resulting sample was HEPES, N-2-hydroxyethylpiperazine-N0 -2-ethanesulfo-
spotted on a MALDI plate twice, and each spot was analyzed nic acid.
twice, resulting in four replicate spectra. The spectra were
analyzed manually, and the peak pairs generated from known References
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