Protein identification via MALDI-PSD-ToF MS spectra of on solid phase support SPITC derivatized peptides
The recording of PSD spectra from m/z values of peptide mass fingerprints provides information about the peptides' primary structure and enables the unambiguous identification of proteins from few peptides with significant Mascot ion scores by an analysis of fragmentation patterns. The difficulties of protein identification with MALDI-PSD-ToF MS spectra are low s/n ratios of fragments, incomplete sequence coverage and complex fragmentation patterns as a cause of random charge stabilisation. The protonation of peptides during the MALDI process is mediated by carboxyl groups of the matrix and mainly stabilised at primary amine groups of the N-termini and the Lysine residues. The stabilised charge induces different fragmentations of the same ion. The derivatization of peptides with SPITC binds a negatively charged sulfonic acid to the peptides' N-terminus . This negative charge neutralises the protonation of C-terminal fragments which are no longer detectable in mass spectrometry. Primarily, the PSD spectra of SPITC-derivatised peptides show fragments of the y-series, whose appearance among the N-terminal fragments is promoted by the large electron density around the peptide bond. The derivatization with SPITC enables the detection of complete y-series with good s/n ratios and offers the elucidation of the peptides' primary sequence. The unambiguous mapping of peptides with completely known primary sequences to proteins registered in databases is remarkably simplified and is expressed in increased Mascot scores . However, the application of SPITC derivatization for protein identification from complex mixtures is limited by the reduction of the peptides' signal intensities following N-terminal sulfonation. This degradation of sensitivity could be decreased by an optimization of the reaction conditions. For increased sensitivity the N-terminal sulfonation of peptides bound on C18 solid phase support is preferable compared to an in solution approach since signal intensity is increased and quality of PSD spectra is raised.
Comparative Proteome Analysis of VACV IHD-W infected HEK293 cells
The comparison of the proteome analysis of VACV IHD-W-infected HEK293 cells with non-infected control cells 9 h post infection has enabled the identification of 24 human proteins, whose expression has been regulated by infection, as well as of 3 viral proteins. Since the genome of VACV IHD-W is not available, the identification of viral proteins occurred via sequence homologies of the VACV strains Western Reserve and Copenhagen. Afterwards peptide sequences were derived from spectra of 13 spots representing proteins regulated in their expression, whose database search gained no result. Single-base substitutions among different VACV strains, which led to amino acid replacement, might have resulted in different peptide mass fingerprints. PSD spectra are in correlation to the confidence interval of the m/z values of the precursor ion. Therefore, protein identification via Mascot Search is disabled by mass shifts of precursor ions resulting from an amino acid replacement. De novo sequencing of viral proteins could not be assured because of the polyproteonality of 2D-gel spots. The unambiguously identified differences in the expression profile of HEK 293 cells resulting from VACV IHD-W infection are discussed in the following sections.
Proteins relevant for energy metabolism
The infection of HEK293 cells with active and inactivated Vaccinia Virus IHD-W significantly affects the cells' energy metabolism. The expression of fructose-bisphosphate aldolase A (A-1), which acts as a central enzyme in the glycolysis by catalysing the retro-aldol cleavage of fructose-1,6-bisphosphate into dihydroxyacetone phosphate and glycerinaldehyde, is increased by both types of infection . Also the enhanced expression of the enzyme malate dehydrogenase (A-2) is directly associated with a boost in the energy metabolism, which serves the viruses' need for energy for DNA replication. This enzyme is an integral part of the citric acid cycle by catalysing the oxidation of malate to fumarate . The resulting reduction equivalent NADH/H+ transfers electrons to the electron transport chain which builds up a proton gradient at the inner mitochondrial membrane, resulting in ATP-synthesis catalysed by the H
-transporting two-sector ATPase (A-3, A-4) . A modification of the α-chain precursor of the H
-transporting two-sector ATPase (A-3, A-4), resulting in spot migration, is triggered by both types of infection and has been detected as well as an overexpression of the mtDNA stabilising ATPase family AAA domain containing 3A (A-6) protein, whose existence has just been evidenced at transcript level . Another energy metabolism-related protein whose expression is up-regulated by the VACV IHD-W infection is the βsubunit of the electron transfer flavoprotein (A-5) which acts as a specific electron acceptor for several dehydrogenases, e.g. malate dehydrogenase
(A-2), and transfers them to the respiratory chain .
All changes detected in the human proteome profile caused by the infection which are related to energy metabolism indicate an enhancement of the metabolic rate of the glycolysis as well as of the oxidative phosphorylation in order to fulfil the viruses' energy need for replication.
Proteins associated with gene expression and protein biosynthesis
The expression of the eukaryotic translation initiation factor 4H (eIF-4H) (B-3) is up-regulated by infectious and inactivated VACV IHD-W. The protein eIF-4H stimulates protein biosynthesis, ATP hydrolysis and helicase activity of eIF-4A. Enzymatic investigations show that the affinity of eIF4A to RNA is increased two-fold and helicase activity four-fold by an interaction with eIF-4H . The regulation of eIF-4H may contribute to an enhancement in protein biosynthesis which is required by the virus' need for protein expression.
Further on an increased expression of nuclear protein Hcc-1
(CIP29) (B-4) is denoted in both types of infected HEK293 cells. This protein possesses a binding domain for ss and dsDNA and is postulated to be part of the ribonuclein complex. Interactions with the RNA-helicases DDX39 and BAT 1 are verified which proves the influence of Hcc-1 on the transcription of DNA. The overexpression of Hcc-1 in HEK293 cells is known to decrease the cells' growth rate [21, 22].
Several members of the spliceosome, heterogeneous nuclear ribonucleoprotein B1 (B-5), novel protein similar to small nuclear ribonucleo-protein polypeptide A (B-6), heterogeneous nuclear ribonucleoprotein M (B-7), are modulated in both types of VACV IHD-W-infected HEK2093 cells. This heterogeneous protein complex splices introns of the hnRNA. Since the poxvirus genome has no introns and splicing of mRNA is therefore obsolete, viral effectors may control the cells' protein biosynthesis by interfering with the cellular hnRNA processing [23, 24].
Far upstream element-binding protein 1 (FUBP1) (B-8) stimulates the expression of transcription factor c-myc by binding onto far upstream element (FUSE) upstream of the c-myc promoter . The protein is a known link between the apoptosis cascade and the c-myc oncogene, it further possesses helicase activity for dsDNA. Experiments with transfected cells show that a high FUBP1 expression increases the expression level of c-myc and in this way protects the cell from apoptosis, while caspase-mediated cleavage of FUBP1 induces apoptosis . Both infectious and inactivated VACV IHD-W enhance the expression of FUBP1.
Ewing sarcoma breakpoint region 1(B-9) is a multifunctional protein with essential functions in gene expression, signal transduction, and mRNA transport and processing. Mutations in the protein-expressing gene lead to the development of Ewing sarcomas and other tumours. The protein expression is suppressed after an infection with UV-inactivated as well as with active viruses .
Prefoldin subunit 1(B-10) is part of a heterohexamer chaperon (prefoldin) which binds cytosolic chaperonin (c.CPN) and transports target proteins to the hexamer. It is also involved in the folding of nascent peptides. Gene deletions of prefoldin result in a dysfunction of the actin-tubulin cytoskeleton . Prefoldin subunit 1 is exclusively identified in cells infected with UV-inactivated VACV.
The described alterations caused by the infection are results of the fact that the virus is taking control of the cells' gene expression as well as the protein biosynthesis and so aims for effective expression of viral proteins and the control of the cellular response.
Proteins relevant for apoptosis
Several viruses in general and poxviruses in particular are known to modulate the hosts' apoptosis pathways in order to avoid the antiviral defence mechanism at a cellular level . This results in an altered expression profile of apoptosis-relevant genes caused by viral gene products. During VACV transcription double-stranded RNA (dsRNA) is synthesised, since the virus possesses overlapping genes on both strands of its DNA. The presence of dsRNA activates the dsRNA-dependent serine/threonine kinase (PKR) which phosphorylates the α-subunit of the translation initiation factor eIF-2 which is essential for protein biosynthesis. This phosphorylation inhibits the translation of mRNA and thereby induces apoptosis [30–33]. An altered modification on the γ-subunit of the translation initiation factor eIF-2 (B-1, B-2) caused by the infection was detected by a spot migration in the 2-DE gel the relevance of which has not yet been determined in the literature. The dsRNA-dependent PKR is usually inhibited by nucleophosmin 1 (C-7) which is ubiquitously expressed in human cells and translocates between nucleus and cytoplasm . The downregulation of nucleophosmin 1 (C-7), which can not be detected in cells infected with active VACV IHD-W, suggests that it is a cellular response to the infection for the purpose of anti-viral defence. This immune response is undercut by the product of the VACV E3L gene, the putative double-stranded RNA binding protein
(D-1) which binds dsRNA and inhibits apoptosis induction by preventing PKR from phosporylating the γ-subunit of the translation initiation factor eIF-2 [35, 36]. The E3L gene product has been identified in HEK 293 cells infected with active VACV IHD-W, which is in correlation to proteome analysis of VACV virions that indicate that the putative double-stranded RNA binding protein (D-1) is not present in the virion but is expressed in the early replication phase.
The present proteome analysis has led to the identification of several further apoptosis-relevant proteins. The expression of two isoforms of prohibitin (C-5, C-6) is decreased after infection with active VACV IHD-W. Prohibitin regulates cell division, inhibits DNA synthesis and sensibilises cells for apoptosis by destabilising the mitochondrial membrane . Beyond that, prohibitin enhances the transcription of p53 .
The infection of HEK293 cells with active and inactivated VACV IHD-W inhibits the expression of the protein Guanine nucleotide-binding protein subunit beta 2-like 1 (C-1) which is an acceptor for activated protein kinase C (PKC). PKC is then bound to the cytoskeleton and conveyed to its target proteins, the MARCKS proteins. The receptor is also engaged in the regulation of the SRC kinase which can inhibit apoptosis by a phosphorylation of caspase-8 [39, 40].
The reduced expression of the proteasome subunit alpha type-7-like (C-2) can be seen as a further cellular defence strategy. The proteasome subunit alpha type-7-like (C-2) is part of the proteasome complex which cleaves peptides proteolytically at the amino acids Arg, Phe, Tyr, Leu and Glu . The inhibition of the proteasome in tumour cell lines induces apoptosis.
The phosphatidylethanolamine-binding protein (C-3, C-4) is modified by an infection with active and inactivated VACV IHD-W, which results in an altered pI value. The protein binds to phosphatidylethanolamine which is presented at the surface of apoptotic cells, inhibits the Raf-kinase, prevents the activation of the MAP-Kinase-cascade and the antiapoptotic transcription factor NFκ-B [42, 43]. It is to be postulated that the protein modification is associated with an altered physiological activity.
In this comparative proteome analysis the 14 kDa cell fusion protein
(D-2) is identified in HEK293 cells infected with active VACV IHD-W. Its presence is predicted according to Swissprot . The variola homologue of the A27L gene is known to play an import role in virus penetration by fusing the outermost of the Golgi-derived membranes enveloping the virus with the cells' plasma membrane.
As expected, the major core protein P4a (D-3) of Vaccinia virions is further identified in both infected cell culture approaches .
Peroxiredoxin-2(E-1) is an antioxidative enzyme which reduces hydrogen- and alkyl-peroxides and supports the antiviral activity of CD8(+) T-cells . In contrast to the negative control, protein expression is enhanced by an infection with active and UV-inactivated viruses.
Transgelin-2 (SM22-alpha homologue) (E-2) has not been functionally characterised yet. The protein possesses an actin-binding domain and is overexpressed in cells infected with both infectious and inactivated VACV IHD-W. It seems as if human transgelin is packed into Vaccinia virions and transferred from cell to cell for an unknown reason . It can be hypothesised that it may serve as an anchor for the virus to move along the cytoskeleton.
Sorting nexin 3 (E-3) has a binding domain (PX domain) for phospholipids and transports proteins within cells from organelles to membranes . The protein expression is enhanced after infection of HEK293 cells with active and UV-inactivated VACV IHD-W.
Profilin (E-4) binds actin and participates in the build-up of the cytoskeleton. As a response of extracellular signals, high concentrations of profilin inhibit the Actin-polymerization which is enhanced by low profilin concentrations . Both types of infection increase expression of human profilin, while VACV possesses a profilin homologue by itself.