Targeted Top-down Mass Spectrometry for the Characterization and Tissue-specific Functional Discovery of Crustacean Hyperglycemic Hormones (CHH) and CHH Precursor-Related Peptides in Response to Low pH Stress

Abstract

Crustacean hyperglycemic hormones (CHHs) are a family of neuropeptides that were discovered in multiple tissues in crustaceans, but the function of most isoforms remains unclear. Functional discovery often requires comprehensive qualitative profiling and quantitative analysis. The conventional enzymatic digestion method has several limitations, such as missing posttranslational modification (PTM) information, homology interference and incomplete sequence coverage. Herein, by using a targeted top-down method, facilitated by higher sensitivity instruments and hybrid fragmentation modes, we achieved the characterization of two CHH isoforms from the sinus glands (SG-CHH) and the pericardial organs (PO-CHH) from the Atlantic blue crab, Callinectes sapidus, with improved sequence coverage compared to earlier studies. In this study, both label-free and isotopic labeling approaches were adopted to monitor the response of CHHs and CHH precursor-related peptide (CPRP) under low pH stress. The identical trends of CPRP and CHH expression indicated that CPRP could serve as an ideal probe in tracking the CHH expression level changes, which would greatly simplify the quantitative analysis of large peptides. Furthermore, the distinct patterns of changes in the expression of CHHs in the SG and the PO suggested their tissue-specific functions in the regulation of low pH stress. Ion mobility-mass spectrometry (IM-MS) was also employed in this study to provide conformation analysis of both CHHs and CPRPs from different tissues.

Graphical Abstract

INTRODUCTION

Neuropeptides are endogenous signaling molecules that broadly participate in the regulation of numerous physiological process.^1,2–5 The biosynthesis process usually begins with mRNA of neuropeptide gene being translated into preprohormones, which is followed by a series of enzymatic cleavages, producing signaling peptides, precursor-related peptides and mature, biologically active neuropeptides.⁶ Neuropeptides within the same family share similar structures and highly conserved sequence motifs,^7,8 and some homologous neuropeptides are universal throughout various species.⁹ The decapod crustacean has been utilized extensively in neuropeptidomic studies in our group, focusing on the peptide profiling^10–12 and stress-related function discovery^13–16.

The crustacean hyperglycemic hormone (CHH) superfamily consists of a group of neuropeptides involved in metabolic control, energy homeostasis, molting, osmotic regulation and various biological activities.^17–28 The superfamily can be divided into two distinct sub-groups, on the basis of their precursor and primary structures: type-1, CHH/ITP and type-2 MIH/MOIH/VIH/GIH.^18,25 One of the major differences between the two subtypes is that preprohormones of type-1 peptides contain a CHH precursor related peptide (CPRP), which is absent in that of type-2 peptides.^25,29 The hyperglycemic activity, which is only observed in type-1 peptides, is another aspect to differentiate the two categories.^17,18,25

The CHH peptides (type-1 in CHH superfamily) are normally comprised of around 72 amino acids, containing three intramolecular disulfide bonds formed between six conserved cysteines^22,30. Their hyperglycemic function was first reported in 1944, as a diabetogenic factor in the eyestalk of Atlantic blue crab, Callinectes sapidus and the sand fiddler crab, Uca pugilator.³¹ The first CHH structure was identified in the shore crab, Carcinus maenas, eyestalks in 1989.³² The characterization of the CHH peptides is always challenging due to the large molecular size, low concentration in vivo^17,27 and PTM complexity.^33,34,35 Edman degradation or the cDNA cloning strategies have been well-established to isolate and identify multiple CHH isoforms in various crustacean species.^21,36–42 In recent decades, mass spectrometry (MS) has become an essential and powerful approach in neuropeptidome studies and has been widely adopted in previous CHH analyses^43–45. With the help of high sensitivity and high-resolution instruments, accurate mass and sequence information of neuropeptides could be obtained from a small amount of sample. In addition, the development of electrospray ionization (ESI) broadened the detection mass range by generating multiply charged ions, which highly benefits the study of CHHs. Enzymatic digestion followed by LC-tandem MS bottom-up analysis generates detailed sequence information about pieces of such large peptides, which would further contribute to database matching^32,37. However, the classical tryptic-digested method often causes problems such as incomplete coverage and missing PTM identification.⁴⁶ Additionally, tryptic peptides from highly homologous peptides introduces interference when being assigned to the theoretical sequences. Top-down analysis would be an alternative solution where intact peptides/proteins are detected and fragmented.^47–50Although this method can be informative for characterizing primary structure and modifications of intact molecules, it is also limited by difficulties in producing extensive gas-phase fragmentation ions, especially for those containing disulfide bonds. Usually, both bottom-up and top-down analysis are employed as complementary approaches to result in better coverage of the CHH peptides^35,51.

Initial reports about the CHH peptides were focused on its occurrence in eyestalks, as well as its physiological function in glucose homeostasis.^26,52,53 The studies demonstrated the discovery of CHH isoforms has been further extended to multiple extra-eyestalk regions, such as guts, pericardial organs, thoracic ganglia, etc.^{23,36,42,43,45,54,55} These CHH isoforms, although homologous, differ slightly from each other in their sequences, giving rise to diverse functional roles in physiological modulation and stress-induced regulation; for example, the abundance of CHH in blue crab eyestalk and thoracic ganglia showed different expression patterns throughout the molt cycle.⁴²

CHH precursor-related peptide (CPRP) is C-terminally flanked by CHH in preprohormone.^29,38,39,41 After being enzymatically cleaved, CHH and CPRP were co-localized in neural tissues²³ and then co-released into hemolymph upon stimuli.^43,56 Comparing to CHH, the CPRP exhibits a smaller molecular size, usually consisting of 33–38 amino acids, which is more facile to fragmentation. It is a circulating neurohormone in the crab; however, its function still remains unknown.⁵⁶

Low pH stress is one of the most common stressors that crustaceans encounter due to their living habitat variation. The Atlantic blue crab, Callinectes sapidus, has been reported to be highly influenced when being exposed to an acidified environment^57–59. However, responses of locomotive activities and body fluid pH in the blue crab were observed to exhibit a delay of about 2hrs from the initial exposure to the CO₂ treatment^58,59. The expression level changes of neuropeptides in the blue crab after 2hr incubation in acidified environment have been investigated to reveal potential neuropeptide players involved in modulation of the animal’s adaptation to acidification stimulus¹⁶. However, most of the reported neuropeptides were under 2 kDa. The function of CHHs in the low pH stress related regulation remains uncovered.

In this study, we applied a hybrid fragmentation method in targeted top-down MS analysis to enhance the profiling coverage and minimize experiment time. The expression levels of CHH and CPRP were quantified under low pH stress, contributing to the first report on the tissue-specific functional correlation between CHH and its precursor-related peptide.

METHODS

See Supporting Information for details about chemicals and materials, animal dissections, stress experiment setup, tissue extraction, reductive dimethylation and offline fractionation.

Briefly, pericardial organ and sinus glands were dissected out from the Atlantic blue crab, Callinectes sapidus.⁶⁰ Neuropeptide extraction was performed using acidified methanol to precipitate large proteins. High pH RP-HPLC was applied to isolate the CHHs and CPRPs. In the stress-related study, CO₂ gas was bubbled into the saltwater tank to acidify the water from normal (pH=8.3) to a moderate pH level (7.6–7.8) and air saturation 50% – 60%. The incubation period in low pH environment was 2 hours. Tissues collected from stressed and control animals were subjected to reductive dimethylation for further quantitation analysis.

Mass Spectrometry Analysis

For CHH sequence characterization, tissue extract was reduced by 5 mM dithiothreitol for 1 hour at room temperature and was immediately desalted using C₁₈ Ziptip pipette tips, eluted into 10 μl 50/50/0.2 ACN/H₂O/FA (v/v/v). The samples were always prepared fresh before analysis to avoid the re-formation of disulfide bonds. Top-down MS analysis was performed on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) interfaced with a Dionex Ultimate 3000 UPLC system (Thermo Fisher Scientific). 2 μl purified sample was loaded onto a self-packed column (150 mm length of 1.7 μm C₁₈ with a 3 μm C₁₈ cap) for online separation. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in ACN. A 120 min gradient was used at flow rate of 0.3 μl/min, starting from 97% A and 3% B, increasing to 10% B at 5 min, 55% B at 90 min, 95% B at 92 min (and remaining for 10 min) and then dropping back to 0% at 105 min. The MS/MS analysis was performed in selected ion mode with an inclusion list containing the mass of CHHs, which are selectively fragmented with the following settings selected ion mode (SIM), MS1 resolution, 120 000; Injection time, 250 ms; AGC target, 3e6; isolation window, m/z 6.0; dd-MS², resolution, 60 000; Injection time, 250 ms; AGC target, 1e6. A set of collision energy parameters used in this experiment was listed in Figure S2.

For label free quantitation, tissue extract of control and stressed groups were desalted using C₁₈ Ziptip pipette tips, dried down and resuspended in 10 μl 0.1% FA in water. The purified samples were subjected to online separation with a Waters nano-Acquity Ultra Performance LC system equipped with a self-packed column (same as described earlier) coupled to a Q-Exactive quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). LC gradient was the same as described above. The MS/MS analysis was performed in selected ion mode (SIM) with an inclusion list containing the mass of CHHs and CPRPs, which are selectively fragmented with the following settings: SIM, isolation window, m/z 6.0; resolution, resolution, 70 000; Injection time, 100 ms; AGC target, 3e6; dd-MS2, resolution, 35 000; Injection time, 100 ms; AGC target, 1e6; normalized collision energy, 30.

For the quantitation analysis using isotopic labeling strategy, labeled tissue extract mixture from low pH stress experiment was analyzed by top-down MS method on Q Exactive system in PRM mode. LC setup was identical as earlier section. The MS/MS analysis was performed in parallel reaction monitor (PRM) mode with an inclusion list containing the mass of CHHs, which are selectively fragmented with the following settings: resolution, 70 000; Injection time, 250 ms; AGC target, 1e6; isolation window, m/z 15.0; PRM, Injection time, 100 ms; AGC target, 5e5; normalized collision energy, 30. isolation window was increased here in order to select both heavy- and light-labeled peptides for fragmentation and further quantitation.

Offline HPLC fractions containing the CHHs and CPRPs were isolated and desalted before direct injection onto the Waters Synapt G2 Ion Mobility-Mass Spectrometer (IM-MS). Approximately 5 μL of each sample was loaded onto a home-made nanospray ion source, and a silver wire of 100 μm thickness was inserted into the borosilicate glass needle for high voltage application. Buffer of 10 mM NH₄OAc was used. The MS instrument was run in positive ion mode. Nanospray voltages ranged between 1.0–2.0 kV and the sampling cone was used at 30 V. The MS cone temperature was 75 °C. In typical nanospray experiments, the size of the spray emitter was maintained at ~ 5 μm. The emitters were pulled from borosilicate glass capillaries using a P-2000 laser-based micropipette puller (Sutter Instruments, Novato, CA, USA). All IM-MS data were collected using Waters Synapt G2 instrument (Waters Corp., Manchester, UK) and analyzed in Waters MassLynx V4.1 (Waters Corp., Manchester, UK).

Data Processing

For sequence profiling, averaged tandem mass spectra were deconvoluted by Xtract CI-3.0 Software (Thermo Scientific Inc., Bremen, Germany), using S/N threshold at 1.5 and fit factor of 40%. The resultant spectra were assigned with b/y and c/z ions within 10 ppm error tolerance. ^Cas-SG-CHH contains N-terminus pyro-glutamate and C-terminus amidation. ^Cas-PO-CHH contains N-terminus pyro-glutamate.

For reductive dimethyl labeling, stress-to-control ratio were calculated out of the intensities of quantitative peak pairs in MS² spectra acquired from PRM method. Three biological replicate experiments were carried out and each bio-replicate were repeated for three technical replicates. The quantitative results were evaluated by Student’s t-test. The p-value < 0.05 represents the statistical significance.

For label-free quantitation, XIC peak areas of most abundant charge states of CHHs and CPRPs, which were determined in “sequence profiling” panel, were added up, respectively. Four biological replicate experiments were carried out for ^Cas-PO-CHH and five for ^Cas-SG-CHH. Each bio-replicate was repeated for two technical replicates. The quantitative results were evaluated by Student’s t-test. The p-value < 0.05 represents the statistical significance.

RESULTS AND DISCUSSION

Sequence Profiling of SG-CHH and PO-CHH

The eyestalks were the first source where the existence of a factor that caused hyperglycemia in the crustacean was identified.³¹ The injection of eyestalk extracts back into the crabs resulted in elevation of blood sugar level. Furthermore, the sinus gland (SG) extract increased the in vivo sugar level almost 3-fold higher than the rise induced by SG-removed eyestalk extract, which indicated the primary location of the diabetogenic factor in the sinus gland. As the related studies expanded further, the factor was given the name of Crustacean Hyperglycemic Hormone (CHH). In the Atlantic blue crab, Callinectes sapidus, the cDNA sequences of CHH from the SG (^Cas-SG-CHH) and that from the pericardial organ (^Cas-PO-CHH) were identified using molecular cloning method.^{20,36 Cas-}SG-CHH and its precursor-related peptide (^Cas-SG-CPRP) are significant components in the sinus gland peptidome (Figure 1g), while the dominance is not observed in the pericardial organ (PO) (Figure 1h). The concentration of ^Cas-PO-CHH is about 10⁻¹¹ M, which is 10-fold lower than the counterpart in the sinus gland at 10⁻¹⁰ M.²⁰ In MS analysis, the precursor ions produced by ESI source are 6⁺, 7⁺ and 8⁺ for both SG and PO CHHs (Figure 1c, 1e) and 4⁺, 5⁺ and 6⁺ for their corresponding precursor-related peptides (Figure 1d, 1f).

Figure 1. Characterization of CHH using offline HPLC and MS.

Full MS spectra of ^Cas-SG-CHH (a) and ^Cas-PO-CHH (b) showing the 6 Da mass difference due to the DTT treatment. Most abundant charge states in MS¹ spectra of the CHHs (c and e) and the CPRPs (d and f) using electrospray ionization. High pH HPLC separation of sinus gland extract (g) and pericardial organ tissue extract (h).

Mass spectrometry has been applied to sequence analysis of the CHH by our group previously.^35,51,61 On top of the amino acid sequence study, post-translational modification (PTM) analysis is essential in functional discovery. Top-down approach outperforms the bottom-up method in analyzing PTMs, benefiting from eliminating artificial modifications or information loss during sample preparation. Intact CHH structures were restrained by three disulfide bonds. Both SG and PO extracts were reduced by DTT, however, no alkylation was performed. Due to the large size of CHHs, the alkylation usually tended to be incomplete³⁵, which would cause peak split at MS¹, leading to decreased precursor intensity. The reduction of three sets of disulfide bonds would cause an increment in mass by 6 Da (Figure 1a, 1b) and considerably promoted peptide fragmentation efficiency (Figure S1). Reduced ^Cas-PO-CHH and of ^Cas-SG-CHH were selectively fragmented on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer under multiple collision energy levels (Figure S2). The protonated molecular mass and fragmented ions in representative MS² spectra of both analytes (Figure 2 a–d) were measured at high accuracy, relative to the theoretical molecular mass (Figure 2 e–g). Overall, under the higher-energy collision dissociation (HCD), 54 and 56 peptide bonds were fragmented and consequently 46 out of 72 and 52 out of 71 amino acid residues were covered in the ^Cas-SG- and ^Cas-PO-CHH, respectively (Figure 3a, 3c). Both the fragmentation map and the tandem mass spectra exhibited that the cleavages preferred to occur on both termini, although the structure restriction imposed by the disulfide bonds has been released by DTT treatment and led to expanded conformation (Figure 6b). A hybrid fragmentation method -- electron-transfer/ higher-energy collision dissociation (EThcD) was applied to the characterization of two CHH peptides, generating both b/y ions and c/z ions. In the deconvoluted spectra, more fragment ions were aligned with residues in the middle region of the peptide chains (Figure 2b, 2d). In the earlier MS-based study on ^Cas-SG-CHH³⁵, only 36 residues were identified with on-line top-down method and the combination of on-line and off-line top-down methods could reach the coverage of 54 residues. The bottom-up sequencing method enabled the identification of 58 residues. In our study, 53 residues were covered in a single on-line top-down method (Figure 3b), demonstrating that a much higher fragmentation efficiency (53/72 versus 36/72) was obtained with less time spent on sample preparation and instrument runs. For ^Cas-PO-CHH, almost every peptide bond was cleaved under EThcD, except for the one located next to the N-terminus pyro-glutamate and the one next to one of the six cysteines (Figure 3d). The identified ions were well spread out in the tandem mass spectra from the low mass range to the high mass range, producing a considerably enhanced fragmentation coverage. The top-down sequencing of ^Cas-PO-CHH strengthened the bottom-up result in the previous study and verified that the methionine oxidations found in that work³⁵ were artificial modifications caused by sample preparation rather than endogenous PTMs.

Figure 2. Top-down tandem MS sequence profiling of CHH isoforms.

MS/MS spectra of ^Cas-SG-CHH (a and b) and ^Cas-PO-CHH (c and d) under HCD fragmentation (a and c) and EThcD fragmentation (b and d) acquired on the Fusion Lumos Orbitrap instrument. Zoom-in full MS spectra of protonated CHHs (e and g) and the representative fragment ion (f) with experimental and calculated masses annotated.

Figure 3. Fragmentation map and active labeling sites.

Fragmentation map of ^Cas-SG-CHH (a and b) and ^Cas-PO-CHH (c and d). Reductive dimethyl labeling active sites (lysine residue) in bold font. Identical residues between two homologous isoforms with grey background. ^Cas-SG-CHH contains N-terminus pyro-glutamate (pQ) and C-terminus amidation on Valine (Va). ^Cas-PO-CHH contains N-terminus pyro-glutamate (pQ).

Figure 6. Gas-phase conformational analysis using ion mobility mass spectrometry.

(a) Drift time alignment of ^Cas-SG-CPRP and ^Cas-PO-CPRP at their most abundant charge states. (b) Conformational change of ^Cas-SG-CHH and ^Cas-PO-CHH measured by shift in ion mobility drift time and CCS changes due to the DTT reduction treatment.

Isotopic Reductive Dimethyl Labeling-assisted Quantitation of SG-CHH in Response to Low pH Stress

The pH level drop is a common environmental fluctuation in blue crabs’ aqueous habitat. While encountering the stressful disturbance, crabs usually switch to an anaerobic energy metabolism, i.e., glycolysis, in which CHH plays essential regulatory roles.⁶² To investigate how ^Cas-SG-CHH is involved in the physiological modulation upon low pH stress, the control and stressed (2-hour incubation in acidified water) samples were labeled by formaldehyde-H₂ (light) and formaldehyde-D₂ (heavy), respectively, followed by LC-MS analysis using Q-Exactive Hybrid Quadrupole Orbitrap. With the free N-terminus being blocked, the active sites for reductive dimethylation labeling on the ^Cas-SG-CHH are the two lysine residues (bold font in Figure 3a, 3b), giving rise to 8 Da mass difference in molecular mass. Precursor ions of both channels exhibited great overlap (Figure 4a) due to the isotopic distribution and the small difference in mass-to-charge ratio (m/z), which is less desirable for quantification purpose. Therefore, we adopted the parallel reaction monitoring (PRM) method to simultaneously select light and heavy labeled precursors for HCD fragmentation. In the tandem mass spectra, y-ions containing the isotopic labeled lysine (the 5^th residue on the C-terminus) were considered as quantitative peak pairs (Figure 4c). There were 4 Da mass difference between labeled y-ions and the dynamic range of the labeling method has been demonstrated from 1:0.1 to 1:10 (Figure S3). The stress-to-control ratios of two quantitative peak pairs, y₈²⁺ and y₉²⁺, were averaged to represent the change of ^Cas-SG-CHH expression upon the low pH stress.

Figure 4. Top-down MS quantitation upon low pH stress assisted by reductive dimethyl labeling.

(a) MS¹ spectrum of isotopic formaldehyde labeled ^Cas-SG-CHH. Expression level change of ^Cas-SG-CHH in response to acidification stress (b) calculated from quantitative peak pairs acquired on Q-Exactive Orbitrap in PRM mode (c). n=3. s/c, stress-to-control ratio. Error bar, standard deviation.

As a result, a modest and insignificant drop was observed in ^Cas-SG-CHH as a response to the 2-hour low pH stress (Figure 4b). The well-known functional role of CHH is to cause hyperglycemia, which elevates the level of glucose and provides energy for physiological process against exterior stress.⁶³ CHH is also involved in a negative feedback regulation with glucose.^26,52,64 Accumulation of glucose in the body fluid, i.e., hemolymph, will inhibit the release of CHH.⁶¹ As the need for energy metabolism and consumption of glucose increases, as the condition discussed here, this will trigger CHH secretion⁶⁵ and last for several hours,⁶² which would deplete the CHH concentration in the neuroendocrine tissue.

The expression level change of ^Cas-SG-CHH was in alignment with that of its precursor-related peptide (^Cas-SG-CPRP) induced by the same stress condition, reported in an earlier study.¹⁶ CPRP, as a co-released peptide with CHH, has a shorter amino acid chain, thus a higher fragmentation efficiency in MS analysis. In vivo, the half-life time of CPRP is approximately 60 min, which is much longer than that of mature CHH at only 5–10 min.^17,27,56,62 Therefore, if CPRP was found to secrete simultaneously with CHH and remain aligned in expression level fluctuations when encountering stress, it would be considered as an ideal probe or surrogate to monitor the change of CHH.

Discovery of Tissue-specific Function of CHH and the Relationship to Its Precursor

Different from its counterpart in the SG, ^Cas-PO-CHH contains the only labeling site (lysine residue) next to the cysteine (bold font in Figure 3c, 3d), which hinders the fragmentation and makes the quantitative peak pairs unavailable. One of the alternative options is to couple the dimethyl labeling process with reduction and alkylation. However, we would like to develop a quantitative method which could be ideally applied to analyze CHHs or other large peptides in the body fluid, demanding minimal sample preparation time to avoid enzymatic degradation. Here, targeted top-down detections of both CHH and CPRP were performed. MS¹ accumulated peak areas were calculated for label-free quantitation and MS² spectra were used for identity validation. In order to discover the tissue-specific function of CHH in the PO and SG, the quantitation of ^Cas-SG-CHH and ^Cas-SG-CPRP were repeated using a label-free strategy.

Both ^Cas-SG-CHH and ^Cas-PO-CHH have blocked N-terminus, which is the major isoform. Another minor species with free N-terminus was also observed during the experiment (Figure 5c 5d). The abundance distribution of isoforms with or without the N-terminus pyro-glutamate is a common pattern throughout many other species and the isoforms showed similar biological functions.^37,66 Similar case was also observed for the CPRPs, the isoform with methionine oxidation being the major species (Figure 5a 5b). In this study, both isoforms were utilized for the quantification. As a result, ^Cas-SG-CHH and ^Cas-SG-CPRP responded to the low pH stress in an unnoticeable variation (Figure 5e), while expression levels for both ^Cas-PO-CHH and ^Cas-PO-CPRP dropped significantly (stress to control ratio (s/c) =0.45, p-value=0.018 and s/c=0.60, p-value=0.005, respectively) (Figure 5f). The label-free quantitation results of the ^Cas-SG-CHH, the ^Cas-SG-CPRP and the ^Cas-PO-CPRP were in agreement with the isotopic label-assisted measurements,¹⁶ indicating the minimum variance in run-to-run reproducibility and negligible sample loss in label-free and labeling strategies, respectively. In each tissue, the CPRPs exhibited the same changing trend as the CHHs. Therefore, the CPRPs can serve as an ideal probe in analyzing the in vivo expression level change of the CHHs upon low pH stress, which is often limited by the rapid degradation after secretion and their much bigger size for MS analysis. However, whether the characterization of CPRP as an indicator to CHH can be applied to a diverse set of stress studies, still needs further investigations.

Figure 5. Tissue-specific functional discovery using label-free strategy.

(a-d) Accumulation peak areas in extracted ion chromatogram (XIC) of CPRPs and CHHs. Stress-induced variation of CHHs and their precursors in the SG (e) and the PO (f) after 2-hour incubation at low pH environment. n=5 for ^Cas-SG-CHH and n=4 in ^Cas-PO-CHH. Student’s t-test was applied to evaluate the significance of expression level change. Error bar, standard deviation, asterisk, p-value < 0.05.

A tissue-specific functional role triggered by the acidification stress could be inferred from the different trends observed in the SG and PO. A possible mechanism is that the ^Cas-SG-CHH and ^Cas-SG-CPRP were irrelevant to the physiological modulation caused by low pH stress. However, ^Cas-PO-CHH and its precursor were released and circulated through the body fluid and exerted their regulatory effect on certain targets. It has been reported that the binding sites for ^Cas-PO-CHH are poorly competed for by ^Cas-SG-CHH, indicating their different functional roles and regions.⁶⁷ Thus, it is possible that more ^Cas-PO-CHH were secreted from the tissue to the hemolymph to compensate the consumption during stress accommodation, leading to the significant drop of its abundance in the tissue.

The diverse response to the same environment stressor by the two CHH isoforms could be inferred from their diversity in sequences. ^Cas-SG-CHH and ^Cas-PO-CHH sequences are identical in the first 40 amino acids, while the latter region towards C-termini exhibits great difference (Figure 3).²⁰ The sequence variance did not produce obvious disparity in gas-phase conformations of the intact peptides due to the three conserved disulfide bonds. Nonetheless, it is noted that the ion mobility drift times deviate from each other significantly after DTT reduction (Figure 6b). Similar sequence homology pattern is also observed in many other species^44,45 and is always accompanied with discrepancy in their functions: the PO-CHHs do not initialize hyperglycemia as how the SG-CHHs perform.⁴⁵ Early studies reported that in the blue crab, both PO-CHH and SG-CHH increased in relative abundance under hypoxia stress. After recovery in normal oxygen level, SG-CHH abundance dropped lower than the basal level, while the concentration of PO-CHH was reset to the initial point.²⁰ However, ^Cas-SG-CPRP and ^Cas-PO-CPRP share the identical sequence (RSAEGLGRMGRLLASLKSDTVTPLRGFEGETGHPLE)⁴² and in our study, no gas-phase conformational difference was observed in the two CPRP isoforms at all abundant charge states using ion mobility MS (Figure 6a). It is speculated that the tissue-specific functional roles of neuropeptides occur not only among different members within the same family, but also among identical peptides in different regions.

CONCLUSION

In this study, we applied targeted top-down MS strategies to characterize the crustacean hyperglycemic hormone (CHH) and to discover the functional roles of the CHH and its precursor-related peptide (CPRP) in response to low pH stress. Benefiting from the advanced instrumentation and the hybrid fragmentation method, EThcD, we characterized the ^Cas-SG-CHH with an improved coverage using shorter experiment time, compared to previous studies. The ^Cas-PO-CHH was, for the first time, profiled using top-down method, which validated the results acquired using bottom-up de novo sequencing. The quantitative results of ^Cas-SG-CHH from labeling-assisted and label-free method were in agreement, confirming the reliability of both strategies. After 2-hour incubation in low pH stress environment, ^Cas-SG-CHHs were not obviously altered in expression level, while the abundance of ^Cas-PO-CHHs was significantly reduced, showing their different regulatory roles involved in the physiological process. In each tissue, the concentration changes in the CPRPs aligned with their corresponding CHHs, indicating that the CPRP can act as an ideal quantitative probe in tracking the CHH expression level alteration during the low pH stress. Collectively, this study highlights the qualitative and quantitative analysis of CHHs and CPRPs with enhanced efficiency and accuracy. This targeted top-down strategy can be further applied to investigate CHH isoforms in circulating body fluid and to uncover the comprehensive stress-related functions of the CHHs and CPRPs. The experimental design and strategy reported here can be applied to the study of large signaling peptides in other biological systems in the future.