Apolipoprotein A2 isoforms associated with exocrine pancreatic insufficiency in early chronic pancreatitis.

BACKGROUND AND AIM: Apolipoprotein A2 (apoA2) isoforms have been reported to undergo the aberrant processing in pancreatic cancer and pancreatic risk populations compared with that in healthy subjects. This study aimed to clarify whether apoA2 isoforms were as useful as N-benzoyl-p-aminobenzoic acid (BT-PABA) test for exocrine pancreatic dysfunction markers in patients with early chronic pancreatitis (ECP). METHODS: Fifty consecutive patients with functional dyspepsia with pancreatic enzyme abnormalities (FD-P) (n = 18), with ECP (n = 20), and asymptomatic patients with pancreatic enzyme abnormalities (AP-P) (n = 12) based on the Rome IV classification and the Japan Pancreatic Association were enrolled in this study. The enrolled patients were evaluated using endoscopic ultrasonography and endoscopic ultrasonography elastography. Five pancreatic enzymes were estimated. Pancreatic exocrine function was analyzed using the BT-PABA test. Lighter and heavier apoA2 isoforms, AT and ATQ levels were measured by enzyme-linked immunosorbent assay methods. RESULTS: There were no significant differences in clinical characteristics such as age, gender, body mass index, alcohol consumption and smoking among patients with AP-P, FD-P, and ECP. The BT-PABA test and lighter apoA2 isoform, AT level in the enrolled patients had a significant correlation (P < 0.01). The BT-PABA test in patients with ECP was significantly lower (P = 0.04) than that in AP-P. ApoA2-AT level in patients with ECP was lower than that in AP-P, albeit, insignificantly. Interestingly, apo A2-AT level was significantly (P = 0.041) associated with exocrine pancreatic insufficiency by multiple logistic regression analysis. CONCLUSIONS: ApoA2-AT level is a useful tool to evaluate exocrine pancreatic insufficiency in the early stage of chronic pancreatitis.

ACTOR: a latent Dirichlet model to compare expressed isoform proportions to a reference panel.

The relative proportion of RNA isoforms expressed for a given gene has been associated with disease states in cancer, retinal diseases, and neurological disorders. Examination of relative isoform proportions can help determine biological mechanisms, but such analyses often require a per-gene investigation of splicing patterns. Leveraging large public data sets produced by genomic consortia as a reference, one can compare splicing patterns in a data set of interest with those of a reference panel in which samples are divided into distinct groups, such as tissue of origin, or disease status. We propose A latent Dirichlet model to Compare expressed isoform proportions TO a Reference panel (ACTOR), a latent Dirichlet model with Dirichlet Multinomial observations to compare expressed isoform proportions in a data set to an independent reference panel. We use a variational Bayes procedure to estimate posterior distributions for the group membership of one or more samples. Using the Genotype-Tissue Expression project as a reference data set, we evaluate ACTOR on simulated and real RNA-seq data sets to determine tissue-type classifications of genes. ACTOR is publicly available as an R package at https://github.com/mccabes292/actor.

Differential expression of myosin heavy chain isoforms type II in skeletal muscles of polar and black bears.

In this study, the pattern of myosin heavy chain (MHC) isoforms expression in skeletal muscles of the trunk, forelimb and hindlimb in Polar Bear (PB) Ursus maritimus; American Black Bear (AmBB), Ursus americanus and Asian Black Bear (AsBB), Ursus thibetanus was analysed by immunohistochemistry and SDS-PAGE. Results showed that slow (MHC-I) and fast (MHC-II) isoforms exist in muscles of bears. Type II fibres were classified further into Type IIa and IIx in PB but not in AsBB and AmBB. The distribution of Type I and Type II fibres in the trunk, forelimb and hindlimb varied based on muscle type and animal species. The proportions of Type I fibres formed approximately one-third of muscle composition in PB (trunk, 32.0%; forelimb, 34.7%; hindlimb, 34.5%) and a half in both AsBB and AmBB whereas Type IIa and IIx formed approximately two-third in PB (trunk, 68.0%; forelimb, 65.3%; hindlimb, 65.5%) and a half of Type II in both AmBB and AsBB. PB is a good swimmer, lives in Arctic Ocean on slippery ice catching aquatic mammals such as seals and is larger in size compared to the medium sized AmBB (living in forest) and AsBB (arboreal). The results suggest that in bears, there is greater diversity in MHC isoforms II, being expressed in selected fast contracting skeletal muscles in response to variety of environments, weight bearing and locomotion.

Risk stratification of pancreatic cancer by a blood test for apolipoprotein A2-isoforms.

Though pancreatic cancer is uncommon, with an age-adjusted annual incidence of 12.9 cases per 100,000 person-years, it is considered a refractory cancer due to the mortality of 11.0 per 100,000 person-years. To efficiently identify patients with potentially surgically-curable pancreatic cancer, high-risk individuals (HRIs) for pancreatic cancer should be identified by easily and minimally invasive methods from the general population. We have identified unique processing patterns in the C-terminal amino acids of apolipoprotein A2 homodimer in the blood of patients with pancreatic cancer and in HRIs, and we called them apoA2-isoforms (apoA2-i). We then established an enzyme-linked immunosorbent assay (ELISA) to measure circulating apoA2-i in the blood stream. The diagnostic accuracy of apoA2-i to distinguish pancreatic cancer HRIs was verified by several retrospective studies, blind testing with the National Cancer Institute (NCI) Early Detection Research Network (EDRN), a prospective study with prediagnostic samples organized by the European Prospective Investigation into Cancer and Nutrition (EPIC) study, and the prospective screening study of pancreatic cancer in Kobe.The apoA2-i blood test is a potential biomarker to identify HRIs and the curative stage of pancreatic cancer in the general population.

Screening for phenotypic outliers identifies an unusually low concentration of a ß-lactoglobulin B protein isoform in bovine milk caused by a synonymous SNP.

BACKGROUND: Milk samples from 10,641 dairy cattle were screened by a mass spectrometry method for extreme concentrations of the A or B isoforms of the whey protein, ß-lactoglobulin (BLG), to identify causative genetic variation driving changes in BLG concentration. RESULTS: A cohort of cows, from a single sire family, was identified that produced milk containing a low concentration of the BLG B protein isoform. A genome-wide association study (GWAS) of BLG B protein isoform concentration in milk from AB heterozygous cows, detected a group of highly significant single nucleotide polymorphisms (SNPs) within or close to the BLG gene. Among these was a synonymous G/A variation at position + 78 bp in exon 1 of the BLG gene (chr11:103256256G > A). The effect of the A allele of this SNP (which we named B') on BLG expression was evaluated in a luciferase reporter assay in transfected CHO-K1 and MCF-7 cells. In both cell types, the presence of the B' allele in a plasmid containing the bovine BLG gene from -922 to + 898 bp (relative to the transcription initiation site) resulted in a 60% relative reduction in mRNA expression, compared to the plasmid containing the wild-type B sequence allele. Examination of a mammary RNAseq dataset (n = 391) identified 14 heterozygous carriers of the B' allele which were homozygous for the BLG B protein isoform (BB'). The level of expression of the BLG B' allele was 41.9 ± 1.0% of that of the wild-type BLG B allele. Milk samples from three cows, homozygous for the A allele at chr11:103,256,256 (B'B'), were analysed (HPLC) and showed BLG concentrations of 1.04, 1.26 and 1.83 g/L relative to a mean of 4.84 g/L in milk from 16 herd contemporaries of mixed (A and B) BLG genotypes. The mechanism by which B' downregulates milk BLG concentration remains to be determined. CONCLUSIONS: High-throughput screening and identification of outliers, enabled the discovery of a synonymous G > A mutation in exon 1 of the B allele of the BLG gene (B'), which reduced the milk concentration of ß-lactoglobulin B protein isoform, by more than 50%. Milk from cows carrying the B' allele is expected to have improved processing characteristics, particularly for cheese-making.

SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources.

Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources.

Patterning of Oncogenic Ras Clustering in Live Cells Using Vertically Aligned Nanostructure Arrays.

As a dominant oncogenic protein, Ras is well-known to segregate into clusters on the plasma membrane for activating downstream signaling. However, current technologies for direct measurements of Ras clustering are limited to sophisticated high-resolution techniques like electron microscopy and fluorescence lifetime imaging. To further promote fundamental investigations and the related drug development, we hereby introduce a nanobar-based platform which effectively guides Ras clusters into quantifiable patterns in live cells that is resolvable under conventional microscopy. Major Ras isoforms, K-Ras, H-Ras, and N-Ras, were differentiated, as well as their highly prevalent oncogenic mutants G12V and G13D. Moreover, the isoform specificity and the sensitivity of a Ras inhibitor were successfully characterized on nanobars. We envision that this nanobar-based platform will serve as an effective tool to read Ras clustering on the plasma membrane, enabling a novel avenue both to decipher Ras regulations and to facilitate anti-Ras drug development.

Immunohistochemical comparison of p63 and p40 in head and neck Spindle Cell Carcinoma.

BACKGROUND: Spindle Cell Carcinoma (SpCC) is a rare type of squamous cell carcinoma (SCC) with prominent malignant spindle cell component. This unique biphasic feature on histopathological examination makes its diagnosis problematic. Detection of p63 antigens in SpCC can be helpful however its expression in variousb proliferating soft tissue lesions demands for better marker. METHODS: In this study, histopathologically diagnosed SpCC of head and neck region were considered as cases, and 22 soft tissue sarcomas, reactive lesions and spindle cell lesions of the body were taken as controls. Immunohistochemistry (IHC) was done using Anti-p63 and p40 clone and the results were compared. CK was done for negative cases to prove their epithelial origin. P. value < 0.05 considered statistically significant. RESULTS: Among 22 cases of SpCC, 19 cases showed positive immunoreactivity to p63, and 18 cases for p40. IHC of controls showed no immunoreactivity in any of the sarcomas, reactive lesions or spindle cell lesions. The sensitivity of p63 is 86% while that of p40 is 82%. Specificity of both the markers was 100% CONCLUSION: Though p63 is a slightly (4%) more sensitive marker than p40, percentage of cell positivity for p40 is higher compared to p63. Both of these markers are 100% specific for SpCC.

Predicting protein interaction network perturbation by alternative splicing with semi-supervised learning.

Alternative splicing introduces an additional layer of protein diversity and complexity in regulating cellular functions that can be specific to the tissue and cell type, physiological state of a cell, or disease phenotype. Recent high-throughput experimental studies have illuminated the functional role of splicing events through rewiring protein-protein interactions; however, the extent to which the macromolecular interactions are affected by alternative splicing has yet to be fully understood. In silico methods provide a fast and cheap alternative to interrogating functional characteristics of thousands of alternatively spliced isoforms. Here, we develop an accurate feature-based machine learning approach that predicts whether a protein-protein interaction carried out by a reference isoform is perturbed by an alternatively spliced isoform. Our method, called the alternatively spliced interactions prediction (ALT-IN) tool, is compared with the state-of-the-art PPI prediction tools and shows superior performance, achieving 0.92 in precision and recall values.

Novel Human Insulin Isoforms and Cα-Peptide Product in Islets of Langerhans and Choroid Plexus.

Human insulin (INS) gene diverged from the ancestral genes of invertebrate and mammalian species millions of years ago. We previously found that mouse insulin gene (Ins2) isoforms are expressed in brain choroid plexus (ChP) epithelium cells, where insulin secretion is regulated by serotonin and not by glucose. We further compared human INS isoform expression in postmortem ChP and islets of Langerhans. We uncovered novel INS upstream open reading frame isoforms and their protein products. In addition, we found a novel alternatively spliced isoform that translates to a 74-amino acid (AA) proinsulin containing a shorter 19-AA C-peptide sequence, herein designated Cα-peptide. The middle portion of the conventional C-peptide contains ß-sheet (GQVEL) and hairpin (GGGPG) motifs that are not present in Cα-peptide. Islet amyloid polypeptide (IAPP) is not expressed in ChP, and its amyloid formation was inhibited in vitro more efficiently by Cα-peptide than by C-peptide. Of clinical relevance, the ratio of the 74-AA proinsulin to proconvertase-processed Cα-peptide was significantly increased in islets from type 2 diabetes mellitus autopsy donors. Intriguingly, 100 years after the discovery of insulin, we found that INS isoforms are present in ChP from insulin-deficient autopsy donors.

Changes in Hemoglobin Isoforms in the Peripheral Blood of Rats with Experimental Posthemorrhagic Anemia.

Six hemoglobin isoforms were identified in the peripheral blood of male Wistar rats by PAAG electrophoresis. Erythrocytes can be separated into 6 fractions by fractional centrifugation; the fractions differ by the content of reticulocytes and cells with fetal hemoglobin. Cells in each fraction contain one light and one heavy hemoglobin isoforms, and their combination is specific to each fraction. Changes in the hemoglobin isoforms in the whole blood in case of blood loss can be associated with changes in physical and chemical properties of circulating erythrocytes, as well as with the formation of various cell populations resulting from activated erythropoiesis.

A Cross-Sectional and Longitudinal Study to Define Alarmins and A-SAA Variants as Companion Markers in Early Rheumatoid Arthritis.

Nowadays, in the study of rheumatoid arthritis (RA), more and more interest is directed towards an earlier effective therapeutic intervention and the determination of companion markers for predicting response to therapy with the goal to prevent progressive joint damage, deformities, and functional disability. With the present work, we aimed at quantifying in a cohort of early RA (ERA) patients naïve to DMARD therapy, proteins whose increase was previously found associated with RA: serum amyloid A (A-SAA) and alarmins. Five A-SAA variants (SAA1α, SAA1ß, SAA1γ, SAA2α, and SAA2ß) but also S100A8 and S100A9 proteins were simultaneously quantified in plasma applying a method based on single targeted bottom-up proteomics LC-MS/MS. First, we compared their expression between ERA (n = 100) and healthy subjects (n = 100), then we focused on their trend by monitoring ERA patients naïve to DMARD treatment, 1 year after starting therapy. Only SAA1α and SAA2α levels were increased in ERA patients, and SAA2α appears to mostly mediate the pathological role of A-SAA. Levels of these variants, together with SAA1ß, only decreased under biologic DMARD treatment but not under methotrexate monotherapy. This study highlights the importance to better understand the modulation of expression of these variants in ERA in order to subsequently better characterize their biological function. On the other hand, alarmin expression increased in ERA compared to controls but remained elevated after 12 months of methotrexate or biologic treatment. The work overcomes the concept of considering these proteins as biomarkers for diagnosis, demonstrating that SAA1α, SAA1ß, and SAA2α variants but also S100A8 and S100A9 do not respond to all early treatment in ERA and should be rather considered as companion markers useful to improve the follow-up of treatment response and remission state. Moreover, it suggests that earlier use of biologics in addition to methotrexate may be worth considering.

Characteristics of α2,3-sialyl N-glycosylated PSA as a biomarker for clinically significant prostate cancer in men with elevated PSA level.

BACKGROUND: The presence of glycosylated isoforms of prostate-specific antigen (PSA) in prostate cancer (PC) cells is a potential marker of their aggressiveness. We characterized the origin of α2,3-sialylated prostate-specific antigen (S23PSA) by tissue-based sialylation-related gene expression and studied the performance of S23PSA density (S23PSAD) alone and in combination with multiparametric magnetic resonance imaging (MRI) for the detection of clinically significant prostate cancer in men with elevated PSA. METHODS: Tissue-based quantification of S23PSA and sialyltransferase and sialidase gene expression was evaluated in 71 radical prostatectomy specimens. The diagnostic performance of S23PSAD was studied in 1099 men retrospectively enrolled in a multicenter systematic biopsy (SBx) cohort. We correlated the S23PSAD with Prostate Imaging Reporting and Data System (PI-RADS) scores in 98 men prospectively enrolled in a single-center MRI-targeted biopsy (MRI-TBx) cohort. The primary outcome was the PC-diagnostic performance of the S23PSAD, the secondary outcome was the avoidable biopsy rate of S23PSAD combined with DRE and total PSA (tPSA), and with or without PI-RADS. RESULTS: S23PSA was significantly higher in Gleason pattern 4 and 5 compared with benign prostate tissue. In the retrospective cohort, the performance of S23PSAD for detecting PC was superior to tPSA or PSA density (PSAD) (AUC: 0.7758 vs. 0.6360 and 0.7509, respectively). In the prospective cohort, S23PSAD was superior to tPSA, PSAD, and PI-RADS (AUC: 0.7725 vs. 0.5901, 0.7439 and 0.7305, respectively), and S23PSAD + PI-RADS + DRE + tPSA was superior to DRE + tPSA+PI-RADS with avoidance rate of MRI-TBx (13% vs. 1%) at 30% risk threshold. CONCLUSIONS: The diagnostic performance of S23PSAD was superior to conventional strategies but comparable to mpMRI.

Systematic detection of functional proteoform groups from bottom-up proteomic datasets.

To a large extent functional diversity in cells is achieved by the expansion of molecular complexity beyond that of the coding genome. Various processes create multiple distinct but related proteins per coding gene - so-called proteoforms - that expand the functional capacity of a cell. Evaluating proteoforms from classical bottom-up proteomics datasets, where peptides instead of intact proteoforms are measured, has remained difficult. Here we present COPF, a tool for COrrelation-based functional ProteoForm assessment in bottom-up proteomics data. It leverages the concept of peptide correlation analysis to systematically assign peptides to co-varying proteoform groups. We show applications of COPF to protein complex co-fractionation data as well as to more typical protein abundance vs. sample data matrices, demonstrating the systematic detection of assembly- and tissue-specific proteoform groups, respectively, in either dataset. We envision that the presented approach lays the foundation for a systematic assessment of proteoforms and their functional implications directly from bottom-up proteomic datasets.

Binding characteristics of [18F]PI-2620 distinguish the clinically predicted tau isoform in different tauopathies by PET.

The novel tau-PET tracer [18F]PI-2620 detects the 3/4-repeat-(R)-tauopathy Alzheimer's disease (AD) and the 4R-tauopathies corticobasal syndrome (CBS) and progressive supranuclear palsy (PSP). We determined whether [18F]PI-2620 binding characteristics deriving from non-invasive reference tissue modelling differentiate 3/4R- and 4R-tauopathies. Ten patients with a 3/4R tauopathy (AD continuum) and 29 patients with a 4R tauopathy (CBS, PSP) were evaluated. [18F]PI-2620 PET scans were acquired 0-60 min p.i. and the distribution volume ratio (DVR) was calculated. [18F]PI-2620-positive clusters (DVR ≥ 2.5 SD vs. 11 healthy controls) were evaluated by non-invasive kinetic modelling. R1 (delivery), k2 & k2a (efflux), DVR, 30-60 min standardized-uptake-value-ratios (SUVR30-60) and the linear slope of post-perfusion phase SUVR (9-60 min p.i.) were compared between 3/4R- and 4R-tauopathies. Cortical clusters of 4R-tau cases indicated higher delivery (R1SRTM: 0.92 ± 0.21 vs. 0.83 ± 0.10, p = 0.0007), higher efflux (k2SRTM: 0.17/min ±0.21/min vs. 0.06/min ± 0.07/min, p < 0.0001), lower DVR (1.1 ± 0.1 vs. 1.4 ± 0.2, p < 0.0001), lower SUVR30-60 (1.3 ± 0.2 vs. 1.8 ± 0.3, p < 0.0001) and flatter slopes of the post-perfusion phase (slope9-60: 0.006/min ± 0.007/min vs. 0.016/min ± 0.008/min, p < 0.0001) when compared to 3/4R-tau cases. [18F]PI-2620 binding characteristics in cortical regions differentiate 3/4R- and 4R-tauopathies. Higher tracer clearance indicates less stable binding in 4R tauopathies when compared to 3/4R-tauopathies.

QuickIsoSeq for Isoform Quantification in Large-Scale RNA Sequencing.

RNA-sequencing (RNA-seq) is a powerful technology for transcriptome profiling. While most RNA-seq projects focus on gene-level quantification and analysis, there is growing evidence that most mammalian genes are alternatively spliced to generate different isoforms that can be subsequently translated to protein molecules with diverse or even opposing biological functions. Quantifying the expression levels of these isoforms is key to understanding the genes biological functions in healthy tissues and the progression of diseases. Among open source tools developed for isoform quantification, Salmon, Kallisto, and RSEM are recommended based upon previous systematic evaluation of these tools using both experimental and simulated RNA-seq datasets. However, isoform quantification in practical RNA-seq data analysis needs to deal with many QC issues, such as the abundance of rRNAs in mRNA-seq, the efficiency of globin RNA depletion in whole blood samples, and potential sample swapping. To overcome these practical challenges, QuickIsoSeq was developed for large-scale RNA-seq isoform quantification along with QC. In this chapter, we describe the pipeline and detailed the steps required to deploy and use it to analyze RNA-seq datasets in practice. The QuickIsoSeq package can be downloaded from https://github.com/shanrongzhao/QuickIsoSeq.

Transcript Isoform-Specific Estimation of Poly(A) Tail Length by Nanopore Sequencing of Native RNA.

The poly(A) tail is a homopolymeric stretch of adenosine at the 3'-end of mature RNA transcripts and its length plays an important role in nuclear export, stability, and translational regulation of mRNA. Existing techniques for genome-wide estimation of poly(A) tail length are based on short-read sequencing. These methods are limited because they sequence a synthetic DNA copy of mRNA instead of the native transcripts. Furthermore, they can identify only a short segment of the transcript proximal to the poly(A) tail which makes it difficult to assign the measured poly(A) length uniquely to a single transcript isoform. With the introduction of native RNA sequencing by Oxford Nanopore Technologies, it is now possible to sequence full-length native RNA. A single long read contains both the transcript and the associated poly(A) tail, thereby making transcriptome-wide isoform-specific poly(A) tail length assessment feasible. We developed tailfindr-an R-based package for estimating poly(A) tail length from Oxford Nanopore sequencing data. In this chapter, we describe in detail the pipeline for transcript isoform-specific poly(A) tail profiling based on native RNA Nanopore sequencing-from library preparation to downstream data analysis with tailfindr.

Transferrin isoform analysis from dried blood spots and serum samples by gel isoelectric focusing for screening congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are a growing, heterogeneous group of genetic disorders caused by a defect in the glycoprotein synthesis. The first and still widely used method for routine CDG screening was isoelectric focusing (IEF) of serum transferrin. Dried blood spot (DBS) testing is commonly used in newborn screening procedures to detect inborn errors of metabolism. The aim of this study was to demonstrate the reliability of the IEF method in DBS testing. Dried blood spot testing can help in the postmortem diagnosis of CDG disorders when other material is unavailable. The patterns and concentrations of transferrin isoforms in serum and DBS are comparable, and slight differences do not affect interpretation of results.

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.

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 post-translational 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.