Shotgun proteomic analysis of wing discs from the domesticated silkworm (Bombyx mori) during metamorphosis

Proteomic profiles from the wing discs of silkworms at the larval, pupal, and adult moth stages were determined using shotgun proteomics and MS sequencing. We identified 241, 218, and 223 proteins from the larval, pupal, and adult moth stages, respectively, of which 139 were shared by all three stages. In addition, there were 55, 37, and 43 specific proteins identified at the larval, pupal, and adult moth stages, respectively. More metabolic enzymes were identified among the specific proteins expressed in the wing disc of larvae compared with pupae and moths. The identification of FKBP45 and the chitinase-like protein EN03 as two proteins solely expressed at the larval stage indicate these two proteins may be involved in the immunological functions of larvae. The myosin heavy chain was identified in the pupal wing disc, suggesting its involvement in the formation of wing muscle. Some proteins, such as proteasome alpha 3 subunits and ribosomal proteins, specifically identified from the moth stage may be involved in the degradation of old cuticle proteins and new cuticle protein synthesis. Gene ontology analysis of proteins specific to each of these three stages enabled their association with cellular component, molecular function, and biological process categories. The analysis of similarities and differences in these identified proteins will greatly further our understanding of wing disc development in silkworm and other insects.     

Characteristics and evolution of the PUF gene family in Bombyx mori and 27 other species

The Pumilio protein is the founding member of the PUF family of RNA-binding proteins, which contains 8 repeat Puf domains and plays important roles during embryogenesis and post-embryogenesis by binding the Nanos response element (NRE) of specific target genes in eukaryotes. In addition, many other proteins containing the Puf domain were identified but with different functions from the Pumilio protein in various species. Taking advantage of the newly assembled genome sequences, in this study we performed a genome-wide analysis of PUF genes in silkworm and other 27 species. In the silkworm, three PUF genes were identified, named Bmpumilio, Bmpenguin and Bmnop by homology analysis. In fungi and animals, four evolutionarily conservational PUF gene families were identified, Group-A, -B, -C and -D. While Group-A, -C, and -D are present in all fungi and animals, Group-B was only identified in fungi. Interestingly, the number and features of the Puf domains are distinct in each group, suggesting different roles for these proteins in every group. The EST and microarray data showed that the mRNA of the three PUF genes can be widely detected in all tissues of the silkworm. Our results provide some new insights into the functions and evolutionary characteristics of PUF proteins.     

Proteomic analysis of rice leaf, stem and root tissues during growth course

Rice proteins were isolated from leaf, stem and root tissues, harvesting at 1, 2, 4, 8 and 10 weeks after budding. Each tissue of each age was separately pulverized in liquid nitrogen, and the resulted tissue powders were suspended in 10% TCA-acetone and followed by acetone suspension to precipitate at low temperature, which resulted in the tissue-specific and age-specific protein mixture. The protein mixtures were separated by 2-DE using polyacrylamide gels (26 x 20 cm). The protein spots were identified by N-terminal sequence analysis and by MALDI and LC-MS/MS analyses after in-gel tryptic digestion. From a total of 4532 spots, 676 unique proteins were identified, of which 80 proteins (12%) were observed in all three tissues: leaf, stem and root. In addition, 45 (7%) were common in leaf and stem, 57 (8%) in stem and root, and 10 (2%) proteins in root and leaf. Also 141 unique proteins (21%) were observed only for leaf, 96 (14%) for stem, and 247 (36%) for root tissue. Proteins playing a role for photosynthesis and energy production were most abundant in leaf and stem, and those for cell defense were rich in roots.     

Functions and substrates of NEDDylation during cell cycle in the silkworm,Bombyx mori

NEDDylation, a post-translational modification mediated by the conjugation of the ubiquitin-like protein Nedd8 to specific substrates, is an essential biological process that regulates cell cycle progression in eukaryotes. Here, we report the conservation of NEDDylation machinery and NEDDylated proteins in the silkworm, Bombyx mori. We have identified all the components necessary for reversible NEDDylation in the silkworm including Nedd8, E1, E2, E3, and deNEDDylation enzymes. By the approach of RNAi-mediated gene silencing, it was shown that knockdown of BmNedd8 and the conjugating enzymes decreased the global level of NEDDylation, while knockdown of deNEDDylation enzymes increased the prevalence of this modification in cultured silkworm cells. Moreover, the lack of the NEDDylation system caused cell cycle arrest at the G2/M phase and resulted in defects in chromosome congression and segregation. Using the wild-type and mutants of BmNedd8, we identified the specific substrates of BmNedd8, which are involved in the regulation for many cellular processes, including ribosome biogenesis, spliceosome structure, spindle formation, metabolism, and RNA biogenesis. This clearly demonstrates that the NEDDylation system is able to control multiple pathways in the silkworm. Altogether, the information on the functions and substrates of the NEDDylation system presented here could provide a basis for future investigations of protein NEDDylation and its regulatory mechanism on cell cycle progression in the silkworm.     

A comparative proteomic analysis of parthenogenetic lines and amphigenetic lines of silkworm

Parthenogenetic strains of silkworm serve as an effective system for sex-control in silkworms. To determine the molecular mechanism of silkworm parthenogenesis, protein profiles from newly hatched silkworm of a parthenogenetic lines with high pigmentation rate and hatching rate were compared with amphigenetic lines using proteomics approach, including by two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS), and bioinformatics analysis. Several proteins were expressed differentially between the parthenogenetic and amphigenetic lines, and seven of nine interesting proteins were identified successfully using MALDI-TOF/TOF MS analysis. The identified proteins were muscular protein-20, odorant binding protein-LOC100301497, glutathione S-transferase delta, translationally controlled tumor protein homolog, cuticular protein RR-1 motif 19, beta-actin, actins, and muscle-type A₁ actins. These proteins may be associated with the regulation of growth, development, and reproductive processes of silkworm parthenogenetic lines.     

家蚕中肠组织抗核型多角体病毒病的相关蛋白分析

家蚕中肠上皮是病毒经口侵入遇到的第一个组织。昆虫幼虫抵御杆状病毒的感染,可通过选择性的使感染的中肠上皮细胞发生调亡并在释放病毒粒子进入血淋巴之前使感染的细胞从中肠脱落。为研究家蚕抗核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)病的机制,通过对BmNPV高度抗性和高度敏感性的家蚕品系杂交和回交构建了近等基因系。本文对家蚕高抗,敏感及近等基因系5龄起蚕中肠组织的蛋白质表达谱进行了二维电泳 (two-dimensional gel electrophoresis,2-DE) 分析,并利用基质辅助激光解吸电离飞行时间 (matrix-assisted laser desorption/ionization-time of flight, MALDI-TOF) 质谱对差异蛋白进行鉴定。结果发现了5个差异表达的蛋白。推测这些蛋白可能与家蚕中肠对BmNPV的抗性或感性有关。     

家蚕对浓核病毒中国株(BmDNV-3)抗性及感性品系中肠的差异蛋白质分析

【目的】通过比较家蚕Bombyx mori抗性及感性品系的中肠蛋白质表达谱,获得家蚕对家蚕浓核病毒中国株（BmDNV-3）抗性相关的蛋白。【方法】利用双向电泳(2-DE)对感性品种华八35和抗性品种秋丰接种病毒后48 h的蛋白质表达谱进行比较分析,并对其中的差异蛋白进行MALDI-TOF-TOF质谱分析,通过NCBInr和MSDB数据库进行蛋白点的鉴定和功能分析。【结果】获得重复性较好的差异蛋白点16个,其中质谱鉴定出5种蛋白,它们分别是糖基转移酶（glycosyltransferase）、糖基转移酶-S（GlcAT-S）、21.5 kD小热休克蛋白（21.5 kD small heat shock protein）、V-ATP酶（vacuolar ATP synthase）和精氨酸激酶（arginine kinase）。这5种蛋白在抗性品系秋丰中的表达量均高于感性品系华八35。【结论】糖基转移酶和糖基转移酶-S仅在抗性品系中存在,提示它们可能是与抗性有关的蛋白。此外,增强的应激反应和能量代谢也可能与家蚕对BmDNV-3的抗性产生相关。     

家蚕脂肪体蛋白质组学研究

通过高精度的双向电泳技术对家蚕末龄幼虫的脂肪体组织进行了研究,采用基质辅助质量飞行时间质谱对其中一些表达量较高的蛋白点进行了鉴定,并利用GPMAW软件结合家蚕基因组预测的蛋白质数据库构建了本地的肽质量指纹图谱数据库,对所得到的肽质量指纹图谱进行了分析。研究发现,经过双向凝胶电泳及其图象分析技术,银染可以分离出722个清晰蛋白点,这些蛋白质主要集中在分子量15～90kD区域,等电点pH4～8之间。MALDI-TOF-MS鉴定的41个蛋白点中都有较强的肽质量指纹信号峰,其中34个蛋白点得到了成功鉴定,其中包括了大量参与代谢的酶类、不同分子量的热激蛋白、重要的血液蛋白30K,Actin3等,这一结果对人们进一步认识家蚕脂肪体提供了有利的帮助。     

Proteome profile of spinneret from the silkworm,Bombyx mori

The silkworm spinneret is an important tissue for silk fibrillogenesis and spinning. All biochemical processes during silk fibrillogenesis are correlated with silk properties. Understanding the role of spinneret in silk fibrillogenesis may help to reveal the mechanism of silk fibrillogenesis as well as improve silk quality for commercial purposes. Thus, we profiled the proteome of silkworm spinneret. A total of 1572 proteins and 232 differential abundance proteins were identified. Silk fibrillogenesis‐related proteins, such as cuticle proteins, ion‐transporting proteins, muscular proteins, and energy metabolic proteins, were abundant in spinneret. Metabolic pathway and GO enrichment analyses revealed that the identified proteins were involved in energy metabolism, chitin binding, and cuticle construction. Active energy metabolism may provide abundant energy for the muscle contraction as well as ion and water exchange. The chitin binding and cuticle construction process may provide sufficient shear forces for silk formation. Our data suggest that silkworm spinneret provides a suitable physiological and biochemical environment for silk fibrillogenesis. These proteins are potential targets for improving silk quality in the silk industry. Data are available via ProteomeXchange with identifier PXD004455.     

The cuticular proteins from free-living and parasitic stages of Haemonchus contortus--I. Isolation and partial characterization

1. Cuticles were isolated from the adult males, adult females, the second molt (2M) sheath from the infective larvae (L3(2M)), and the parasitic third stage (L3) of the sheep parasite Haemonchus contortus by a combination of mechanical disruption and detergent treatment. 2. The 2ME soluble cuticular proteins from adult males contained 4 or 5 major protein bands with molecular weights ranging from 100 to 56 kD with the most prominent band at 56 kD. The cuticular proteins from adult females were similar to the male. 3. Cuticular proteins from the larval stages, 2M cuticle, and L3 cuticle, differed from the adults and from each other. The most prominent protein bands were observed with molecular weights on 78 and 39 kD for the L3 cuticle and 100, 91 and 46 kD for the 2M cuticle. The 2ME soluble cuticular proteins from all developmental stages were at least partially digested by bacterial collagenase. 4. The amino acid composition of cuticular proteins was similar for the L3 and 2M, but adults had lesser amounts of glycine and greater amounts of basic amino acids than the larval stages. The amount of the isolated cuticle solubilized by the 2ME treatment was greatest in adults (80%) compared to the L3 (64%) and the 2M (22%). 5. These results support a hypothesis that there are quantitative and qualitative stage specific differences in the cuticular proteins of H. contortus.     

The cuticular proteins of Tenebrio molitor: II. Patterns of synthesis during postembryonic development

The program of synthesis for the soluble cuticular proteins of Tenebrio molitor was determined by following the incorporation of labeled leucine after a 4-hr pulse in vivo. Soluble proteins were extracted from labeled cuticles and separated on SDS-polyacrylamide slab gels; individual gel slices were counted. The synthetic patterns of larvae and pupae were similar to one another but distinct from the adult pattern. At each stage, the preecdysial pattern was unlike that of postecdysial animals. Distinct periods of synthesis were detected for different proteins. One protein was synthesized and deposited throughout cuticle formation in all three metamorphic stages. One group was synthesized only after ecdysis, while synthesis and secretion of other proteins were restricted to the preecdysial period. Some cuticular proteins never acquired detectable label.     

家蚕幼虫5龄期中肠蛋白质组学

利用双向电泳技术对家蚕幼虫5龄期第2天、第5天和第7天的中肠蛋白质进行分离, 并利用ImageMaster 2D Platinum对所分离得到的蛋白图谱进行比较, 并对一些蛋白质斑点进行了质谱鉴定。研究发现, 家蚕中肠蛋白质具有区别于家蚕其他组织的明显特征: 蛋白质大多分布在PI值4-8、分子量20~70 kD的区域, 且分布不均匀, 主要集中在酸性一侧, 这一特点在家蚕5龄期第7天的图谱尤为明显。5龄期家蚕第2天的蛋白质斑点数目为869个, 而到第5天增加到966个, 新增蛋白数目97个, 进一步观察发现增加的蛋白主要分布在PI值6-9, 分子量20~40 kD区间; 随着进入幼虫成熟期, 蛋白质斑点数目明显减少, 第7天斑点数仅为420个, 比第5天减少了56.5%。这些结果说明家蚕中肠组织蛋白质组成在5龄早、中、晚期经历了较大变化, 暗示这可能与中肠的功能相适应。从MALDI-TOF-MS鉴定的斑点发现了构成家蚕中肠组织的一些新的部分结构蛋白和一些可能与消化、吸收相关的蛋白, 还发现一些能够抵御外界微生物入侵的相关蛋白。这些结果为进一步认识家蚕中肠提供了重要的理论基础。     

沙葱萤叶甲表皮蛋白基因的鉴定及表达谱分析

【目的】表皮蛋白是昆虫体壁的主要组成部分,在昆虫生长发育中起着重要的作用。本研究旨在鉴定沙葱萤叶甲Galeruca daurica表皮蛋白基因,分析其表达模式,以期为进一步研究其在沙葱萤叶甲生长发育中的作用提供必要的基础。【方法】根据本实验室组装的沙葱萤叶甲转录组测序数据,应用生物信息学方法鉴定表皮蛋白基因全长开放阅读框(ORF);采用RT-qPCR技术测定鉴定的8个表皮蛋白基因在沙葱萤叶甲不同发育阶段和3龄幼虫不同组织(头部、体壁、消化道和脂肪体)中的表达谱。【结果】基于转录组数据鉴定到8条沙葱萤叶甲表皮蛋白基因的开放阅读框(ORF)全长序列,命名为GdauCP1-8(GenBank登录号: MN629000-MN629007),ORF长417~810 bp,编码138~269个氨基酸,预测分子量为15~28 kD,等电点pI为4.45~8.62;具有16~20个氨基酸的信号肽;GdauCP1具有典型的跨膜结构,其余7个GdauCP蛋白无跨膜结构。同源序列比对和系统发育分析表明,GdauCP3与马铃薯甲虫Leptinotarsa decemlineata CP的氨基酸序列一致性最高,为60.00%;其余的GdauCPs与玉米根萤叶甲Diabrotica virgifera virgifera CP的氨基酸序列一致性最高,为58.52%~80.00%。GdauCP1-4属于RR-2亚家族,GdauCP5-7属于RR-1亚家族,GdauCP8的亚家族归属未确定。RT-qPCR分析表明,8个GdauCP基因在沙葱萤叶甲不同发育阶段及3龄幼虫不同组织内均有表达,且表达量存在显著差异。GdauCP2, GdauCP4, GdauCP5和GdauCP6在1龄幼虫期高表达,GdauCP3, GdauCP7和GdauCP8在蛹期高表达,GdauCP1在3龄第3天幼虫期高表达;除GdauCP2在成虫中表达水平较高外,其他GdauCP基因在成虫中的表达水平均很低。GdauCP1在头部和体壁中高表达,GdauCP2和GdauCP8在脂肪体中高表达,GdauCP3, GdauCP4, GdauCP6和GdauCP7在消化道中高表达,而GdauCP5在体壁中高表达。【结论】8个GdauCP基因在沙葱萤叶甲不同发育阶段和组织间差异表达,且表达模式不同,意味着不同GdauCP可能具有不同的功能。     

Mass spectrometrical analysis of cuticular proteins from the wing of Hebemoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)

Although several insect cuticular genes and proteins are annotated and an arthropod cuticular database is available, mass spectrometrical data on cuticular proteins and their post-translational modifications are limited. Wings from Hebemoia glaucippe were analyzed by scanning electron microscopy or homogenized, proteins were extracted and run on 2DE. In-gel digestion was carried out by using trypsin, chymotrypsin and Asp-N and subsequently the resulting peptides and post-translational modifications were identified by ion trap tandem mass spectrometry (nano-LC-ESI-MS/MS; HCT). A complex wing skeleton and the cuticle of H. glaucippe were demonstrated. Cuticle protein 18.6, isoform A, pupal cuticle protein, cuticular protein CPR59A and two putative proteins, putative cuticular protein B2DBJ and putative cuticle protein CPG31 with two expression forms were identified. Two phosphorylation sites on the same peptide, T213 and S214, were identified on putative cuticle protein CPG31, quinone formation was observed at Y76 on cuticular protein CPR59A probably indicating the presence of post-translational modifications. The results may be relevant for the interpretation of mechanoelastic and physical properties of these proteins. Along with the extraordinary architecture the proteinaceous matrix is probably representing or allowing the unusual aerodynamic function of the butterfly wing. Moreover, the results may be important for mechanisms of insecticide and drought resistance.     

2‐D PAGE and MS analysis of proteins from formalin‐fixed,paraffin‐embedded tissues

In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin‐fixed, paraffin‐embedded (FFPE) tissues. However, 2‐D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2‐D PAGE separation and MS identification of full‐length proteins extracted from FFPE skeletal muscle tissue. The 2‐D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2‐D maps of proteins from FFPE tissue following standard mass‐compatible silver staining. Protein spots from both FFPE and frozen tissue 2‐D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2‐D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh‐frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full‐length proteins from FFPE tissues might be suitable to 2‐D PAGE‐MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological‐fixed tissues.     

Proteomic analysis of the immune response of the silkworm infected by Escherichia coli and Bacillus bombyseptieus

Abstract The silkworm, Bombyx mori, is an economically important insect with a 5 000‐year history of domestication. During evolution, the silkworm has developed highly effective defenses against invasion and parasitization by microorganisms. In this study, two microorganisms Escherichia coli and Bacillus bombyseptieus were orally infected to silkworm larvae. After infection with E. coli and B. bombyseptieus for 24 h, we investigated the polypeptide changes in the hemolymph, midgut and integument using two‐dimensional gel electrophoresis and matrix‐assisted laser desorption ionization time of flight mass spectrometry. Forty‐seven differentially expressed proteins were identified in these tissues. They belonged to a variety of functional classes, including immune proteins, metabolic proteins and structural proteins. Compared with controls, E. coli‐infected silkworms showed 21 up‐regulated proteins, 25 down‐regulated proteins and lost one protein. After infection with B. bombyseptieus, silkworms showed 15 up‐regulated proteins, 27 down‐regulated proteins, lost three proteins and retained two proteins unchanged. We speculate that all these proteins may play a role in the silkworm immune response, although it is unclear why and how the two kinds of bacteria can so markedly alter expression of these proteins. These results offer valuable insights for measuring the proteomic responses of the silkworm innate immune mechanism.     

Genome‐wide identification of chitin‐binding proteins and characterization of BmCBP1 in the silkworm,Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.     

Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus craniifer

Proteins were extracted from the cuticle of mid-instar nymphs of locusts, Locusta migratoria, and cockroaches, Blaberus craniifer. Seven proteins were purified from the locust extract and five from the cockroach extract, and their amino acid sequences were determined. Polyacrylamide gel electrophoresis indicates that the proteins are present only in the post-ecdysially deposited layer of the nymphal cuticles. One of the locust and one of the cockroach nymphal proteins contain a 68-residue motif, the RR-2 sequence, which has been reported for several proteins from the solid cuticles of other insect species. Two of the cockroach proteins contain a 75-residue motif, which is also present in a protein from the larval/pupal cuticle of a beetle, Tenebrio molitor, and in proteins from the exoskeletons of a lobster, Homarus americanus, and a spider, Araneus diadematus. The motif contains a variant of the Rebers-Riddiford consensus sequence, and is called the RR-3 motif. One of the locust and three of the cockroach post-ecdysial proteins contain one or more copies of an 18-residue motif, previously reported in a protein from Bombyx mori pupal cuticle. The nymphal post-ecdysial proteins from both species have features in common with pre-ecdysial proteins (pharate proteins) in cuticles destined to be sclerotised; they show little similarity to the post-ecdysial cuticular proteins from adult locusts or to proteins from soft, pliable cuticles. Possible roles for post-ecdysial cuticular proteins are discussed in relation to the reported structures.     

Comparative Proteome Analysis Reveals that Cuticular Proteins Analogous to Peritrophin‐Motif Proteins are Involved in the Regeneration of Chitin Layer in the Silk Gland of Bombyx mori at the Molting Stage

The silk gland of silkworm produces silk proteins during larval development. Many studies have long focused on the silk gland of the fifth instar larvae, but few have investigated this gland at other larval stages. In the present study, the silk gland proteomes of the fourth instar and fourth molt are analyzed using liquid chromatography–tandem mass spectrometry. In total, 2654 proteins are identified from the silk gland. A high abundance of ribosomal proteins and RR‐motif chitin‐binding proteins is identified during day 2 of the fourth instar (IV‐2) larval developmental stage, and the expression of cuticular proteins analogous to peritrophin (CPAP)‐motif chitin‐binding proteins is higher during the fourth molt (IV‐M). In all, nine enzymes are found to be involved in the chitin regeneration pathway in the silk gland. Among them, two chitinase and two chitin deacetylases are identified as CPAP‐motif proteins. Furthermore, the expression of CPAP3‐G, the most abundant CPAP‐motif cuticular protein in the silk gland during the IV‐M stage, is investigated using western blot and immunofluorescence analyses; CPAP3‐G shows a reverse changing trend with chitin in the silk gland. The findings of this study suggest that CPAP‐motif chitin‐binding proteins are involved in the degradation of the chitin layer in the silk gland. The data have been deposited to the ProteomeXchange with identifier PXD008677.     

Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.