In-depth analysis of the chicken egg white proteome using an LTQ Orbitrap Velos

The avian egg white functions as a shock-absorber, keeps the yolk in place, constitutes an antimicrobial barrier, and provides water, protein and other nutrients to the developing embryo. Besides these biological roles it is an inexpensive source of high quality protein for food industries, contains proteins of pharmaceutical interest, and proteins that have found widespread use in biomedical research and protein chemistry [1–6]. Therefore, it is no surprise that egg white has been the target of proteomic studies previously. Raikos et al. [7] used 2D electrophoresis to separate the proteins and MALDI-TOF-based peptide mass fingerprinting to analyze the spots. Seven proteins were identified. 2D electrophoresis, peptide mass fingerprinting and LC-MS/MS using a quadrupole-TOF mass spectrometer were used to identify sixteen proteins in a more advanced study [8]. We have reported the high confidence identification of 78 proteins in egg white using a workflow consisting of SDS-PAGE to separate proteins, coupled to LC-MS/MS and MS³ with an LTQ-FT mass spectrometer [9]. The use of combinatorial hexapeptide libraries [10] in conjunction with LC-ESI-IT-MS/MS allowed the identification of 148 egg white proteins, demonstrating the power of this novel technology to detect minor components even in samples dominated by a few major proteins [11]. Bead-coupled peptide libraries are thought to "equalize" the proteome by providing similar numbers of binding sites to each of the different proteins contained in a proteome. However, it was shown recently that, in contrast to the previously proposed mode of action, the beneficial effect of the peptide beads does not appear to be mediated by specific interaction but is instead dominated by simple hydrophobic effects [12].

Samples, such as egg white, where ovalbumin, ovotransferrin and ovomucoid make up approximately 75% of the total protein, are traditionally difficult to analyze in depth by mass spectrometry, because the peptides of these few proteins tend to dominate the full mass spectra and are selected for fragmentation by MS/MS over and over again. This difficulty has been addressed by the above-mentioned peptide ligand library bead or hydrophobic bead technology [10–12]. However, disadvantages of the peptide library technology include that it is only amenable to soluble proteins and that the composition of the proteome is modified in an unknown and unpredictable way, which makes it impossible to determine the absolute quantity of the proteins. Since the publication of those studies, new developments in instrumentation and peptide identification software occurred, which raised the possibility that in-depth investigation of the egg white proteome would not have to rely on enrichment technologies any more. In the present report we used a novel dual pressure linear ion trap instrument, the LTQ Orbitrap Velos [13]. This new generation of mass spectrometers has increased sensitivity and scan speed as compared to the LTQ-FT used in our previous study [9]. The LTQ Orbitrap Velos is fast enough to isolate and fragment ten or more peaks simultaneously with the acquisition of one high resolution mass full scan spectrum. For evaluation of spectra and database searches we used the MaxQuant software, which is particularly suited for the use of high-resolution MS data and yields very high mass accuracy and peptide identification rates [14–16].

Egg white proteins were separated by PAGE and gels were cut into 24 sections for in-gel digestion (Figure 1) followed by mass spectrometric analysis of the resulting peptides on a high resolution instrument with fast sequencing speed. Three repetitions of the experiment resulted in seventy-two raw-files that yielded a total of approximately 61,500 peptides identified and accepted with a peptide posterior error probability (PEP) of <0.01 and a preset false discovery rate (FDR) of 0.01. Of these, 1,373 peptides were sequence-unique. The average absolute mass deviation was 1.2ppm. By searching of a chicken protein sequence database and by accepting only protein identifications with two sequence-unique peptides occurring in at least two of three experimental data sets, 158 proteins were identified (Additional file 1: Egg white proteins identified with two or more unique peptides). If approximately equal conditions are used between the present study and the peptide library-based study [11] by also considering proteins identified with single peptides occurring in at least two experimental data sets, or proteins identified by two or more unique peptides in only one experimental data set, 44 more proteins can be added to the list (Additional file 2: Tentatively identified egg white proteins), resulting in a total of 202 possibly identifications. Additional protein data, such as UniProt and RefSeq accession codes, number of identified peptides, sequence coverage, and protein PEP scores for accepted proteins (without contaminants) are provided in Additional file 3: Protein data. These results compared favorably with those obtained with peptide ligand library beads [11], where 68 proteins were identified with two or more sequence-unique peptides and a total of 148 proteins were obtained by accepting unique single peptide hits from different experiments (Figure 2). Furthermore, our study conservatively groups proteins with very similar sequences together and counts them as one "protein group", even when unique peptides pointed at the presence of isoforms or very similar proteins possibly encoded in different genes. Thus, the number of identified proteins is probably higher. A representative example is ovotransferrin, which seemed to represent a mixture of several forms containing many shared and a few unique peptides. Unique peptide data for accepted proteins (without contaminants), such as sequences, PEP scores, and distribution among gel sections are shown in Additional file 4: Peptide data.

Several previously identified proteins [9] were not identified immediately in the new egg white proteome. However, searching the new database version, IPIchick v3.65, with peptide sequences responsible for the previous identification of these proteins indicated that this was in many cases due to changes in the database. Thus, for instance, the ovosecretoglobulin sequence was no longer joined to a channel protein sequence in IPI00575434 but appeared with a new accession number, IPI00847051. Other proteins changed name. Thus, chondrogenesis-associated lipocalin (IPI00600353) is now lipocalin-type prostaglandin synthase D. The only proteins that could not be identified again in the present study were HMG-1 (IPI00595982), a hypothetical protein (IPI00597019), histone H1 (IPI00597019), 60S ribosomal protein L27 (IPI00577674) and poly(ADP-ribosyl) polymerase 1 (IPI00588387). The first two proteins were previously identified predominantly (HGM-1) or exclusively (Hypothetical protein) by in-solution tryptic cleavage, which was not performed in the present study. Three of these proteins, HMG-1, histone H1, and poly(ADP-ribosyl) polymerase were, however, confirmed in a recent study [11]. Therefore, the reason for their absence in the present study is not clear, but as these proteins are unlikely to play functional roles in egg white, their inclusion in egg white preparations may vary. Keratins were excluded from our results because they usually shared all or most peptides with common contaminants. Only few of the new egg white proteins identified using peptide ligand library beads [11] were also detected in the present study. These were nine proteins in the group of identifications with >2 unique peptides (Additional file 1: Egg white proteins identified with two or more unique peptides) and four among the tentatively identified proteins (Additional file 2: Tentatively identified egg white proteins).

Reassuringly, only two new protein identifications were contained among the 30 most abundant egg white proteins (Additional file 1: Egg white proteins identified with two or more unique peptides). This group of proteins contained 79 proteins that were not identified as egg white components previously. The new egg white proteins included several typical major yolk residents, such as apovitellinin-I, vitellogenin-1 to -3 and apolipoprotein B. These proteins are synthesized in the liver, carried to the ovary via the blood circulation, taken up by oocytes via receptor-mediated processes, and incorporated into the globular fraction of egg yolk [20]. Because the egg yolk was not damaged during mechanical separation of egg white and yolk, these proteins do not seem to be simple contaminants. Rather, residual protein not taken up by the egg cell may be liberated from the ovary together with the egg and migrate with the egg into the oviduct, mixing with egg white proteins secreted in the magnum section. In line with this suggestion, apovitellenin-I and vitellogenins have also been identified in the eggshell organic matrix [21]. This indicates that the oviduct fluid in the eggshell gland still contained these proteins. A few representative peptide fragmentation spectra for some of these proteins are shown in Figure 3. However, many of the new proteins present at low abundance are proteins normally found in intracellular compartments (Additional file 1: Egg white proteins identified with two or more unique peptides; Additional file 2: Tentatively identified egg white proteins). Golgi and ER proteins may have reached the oviduct fluid as by-products of the secretion of major egg white proteins. Other intracellular proteins may have come from damaged, leaky cells of the epithelium lining the oviduct, or from organelles, such as lysosomes, which occur in egg white [22]. Analysis of previously known subcellular locations of proteins identified in egg white shows a decrease in secreted proteins from approximately 64% in the whole proteome to 37% among the new proteins and 18% in tentative identifications (Figure 4). This is accompanied by a similar increase in intracellular proteins, indicating that we have now reached a depth of proteome characterization beyond which it may become difficult to identify functional egg white components. Therefore, minor specific egg white proteins of interest, such as MMP-2, may preferentially be enriched by specific methods before analysis [23]. However, the search for minor components in egg white remains of importance, because very low-abundance proteins, such as bone morphogenetic protein 1 (Additional file 1: Egg white proteins identified with two or more unique peptides) may have a biological role, for instance in early embryonic development.

Our results indicate that current state of the art mass spectrometry technology is sufficiently advanced to permit direct mining of minor components of proteomes dominated by a few major proteins without the necessity to resort to broad specificity protein enrichment techniques, such as peptide ligand library tools, that change the proteome and render absolute quantification impossible. In addition we have significantly expanded the previously known egg white protein inventory.