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proteomics and genomics-1 proteomics and genomics-1 Presentation Transcript

  • www. .uni-rostock.de Bioinformatics Introduction to genomics and proteomics I Ulf Schmitz ulf.schmitz@informatik.uni-rostock.de Bioinformatics and Systems Biology Group www.sbi.informatik.uni-rostock.de Ulf Schmitz, Introduction to genomics and proteomics I 1
  • www. Outline .uni-rostock.de Genomics/Genetics 1. The tree of life • Prokaryotic Genomes – Bacteria – Archaea • Eukaryotic Genomes – Homo sapiens 2. Genes • Expression Data Ulf Schmitz, Introduction to genomics and proteomics I 2
  • Genomics - Definitions www. .uni-rostock.de Genetics: is the science of genes, heredity, and the variation of organisms. Humans began applying knowledge of genetics in prehistory with the domestication and breeding of plants and animals. In modern research, genetics provides tools in the investigation of the function of a particular gene, e.g. analysis of genetic interactions. Genomics: attempts the study of large-scale genetic patterns across the genome for a given species. It deals with the systematic use of genome information to provide answers in biology, medicine, and industry. Genomics has the potential of offering new therapeutic methods for the treatment of some diseases, as well as new diagnostic methods. Major tools and methods related to genomics are bioinformatics, genetic analysis, measurement of gene expression, and determination of gene function. Ulf Schmitz, Introduction to genomics and proteomics I 3
  • Genes • • • www. .uni-rostock.de a gene coding for a protein corresponds to a sequence of nucleotides along one or more regions of a molecule of DNA in species with double stranded DNA (dsDNA), genes may appear on either strand bacterial genes are continuous regions of DNA bacterium: • a string of 3N nucleotides encodes a string of N amino acids • or a string of N nucleotides encodes a structural RNA molecule of N residues eukaryote: • a gene may appear split into separated segments in the DNA • an exon is a stretch of DNA retained in mRNA that the ribosomes translate into protein Ulf Schmitz, Introduction to genomics and proteomics I 4
  • www. Genomics .uni-rostock.de Genome size comparison Species Human (Homo sapiens) Mouse (Mus musculus) Puffer fish (Fugu rubripes) Malaria mosquito (Anopheles gambiae) Fruit Fly (Drosophila melanogaster) Roundworm (C. elegans) Bacterium (E. coli) Chrom. Genes Base pairs 46 28-35,000 3.1 billion 40 22.5-30,000 2.7 billion 44 31,000 365 million 6 14,000 289 million 8 14,000 137 million 12 19,000 97 million 1 5,000 4.1 million (23 pairs) Ulf Schmitz, Introduction to genomics and proteomics I 5
  • www. Genes .uni-rostock.de exon: A section of DNA which carries the coding A section of DNA which carries the coding sequence for a protein or part of it. Exons sequence for a protein or part of it. Exons are separated by intervening, non-coding are separated by intervening, non-coding sequences (called introns). In eukaryotes sequences (called introns). In eukaryotes most genes consist of a number of exons. most genes consist of a number of exons. intron: An intervening section of DNA which occurs An intervening section of DNA which occurs almost exclusively within a eukaryotic gene, but almost exclusively within a eukaryotic gene, but which is not translated to amino-acid sequences in which is not translated to amino-acid sequences in the gene product. the gene product. The introns are removed from the pre-mature The introns are removed from the pre-mature mRNA through a process called splicing, which mRNA through a process called splicing, which leaves the exons untouched, to form an active leaves the exons untouched, to form an active mRNA. mRNA. Ulf Schmitz, Introduction to genomics and proteomics I 6
  • www. Genes .uni-rostock.de Examples of the exon:intron mosaic of genes exon intron Globin gene – 1525 bp: 622 in exons, 893 in introns Ovalbumin gene - ~ 7500 bp: 8 short exons comprising 1859 bp Conalbumin gene - ~ 10,000 bp: 17 short exons comprising ~ 2,200 bp Ulf Schmitz, Introduction to genomics and proteomics I 7
  • Picking out genes in genomes www. .uni-rostock.de • Computer programs for genome analysis identify ORFs (open reading frames) • An ORF begins with an initiation codon ATG (AUG) • An ORF is a potential protein-coding region • There are two approaches to identify protein coding regions… Ulf Schmitz, Introduction to genomics and proteomics I 8
  • Picking out genes in genomes 1. • • • 2. • • • www. .uni-rostock.de Detection of regions similar to known coding regions from other organisms Regions may encode amino acid sequences similar to known proteins Or may be similar to ESTs (correspond to genes known to be expressed) Few hundred initial bases of cDNA are sequenced to identify a gene Ab initio methods, seek to identify genes from the properties of the DNA sequence itself Bacterial genes are easy to identify, because they are contiguous They have no introns and the space between genes is small Identification of exons in higher organisms is a problem, assembling them another… Ulf Schmitz, Introduction to genomics and proteomics I 9
  • Picking out genes in genomes www. .uni-rostock.de Ab initio gene identification in eukaryotic genomes • The initial (5´) exon starts with a transcription start point, preceded by a core promoter site such as the TATA box (~30bp upstream) – Free of stop codons – End immediately before a GT splice-signal binds and directs RNA polymerase to the correct transcriptional start site Ulf Schmitz, Introduction to genomics and proteomics I 10
  • Picking out genes in genomes www. .uni-rostock.de 5' splice signal 3' splice signal Ulf Schmitz, Introduction to genomics and proteomics I 11
  • Picking out genes in genomes www. .uni-rostock.de Ab initio gene identification in eukaryotic genomes • Internal exons are free of stop codons too – Begin after an AG splice signal – End before a GT splice signal Ulf Schmitz, Introduction to genomics and proteomics I 12
  • Picking out genes in genomes www. .uni-rostock.de Ab initio gene identification in eukaryotic genomes • The final (3´) exon starts after a an AG splice signal – Ends with a stop codon (TAA,TAG,TGA) – Followed by a polyadenylation signal sequence Ulf Schmitz, Introduction to genomics and proteomics I 13
  • www. .uni-rostock.de Humans have spliced genes… Ulf Schmitz, Introduction to genomics and proteomics I 14
  • DNA makes RNA makes Protein Ulf Schmitz, Introduction to genomics and proteomics I www. .uni-rostock.de 15
  • www. Prokaryotes Tree of life .uni-rostock.de Ulf Schmitz, Introduction to genomics and proteomics I 16
  • Genomics – Prokaryotes • – • • .uni-rostock.de the genome of a prokaryote comes as a single double-stranded DNA molecule in ring-form – – • www. in average 2mm long whereas the cells diameter is only 0.001mm < 5 Mb prokaryotic cells can have plasmids as well (see next slide) protein coding regions have no introns little non-coding DNA compared to eukaryotes – in E.coli only 11% Ulf Schmitz, Introduction to genomics and proteomics I 17
  • Genomics - Plasmids www. .uni-rostock.de • Plasmids are circular double stranded DNA molecules that are separate from the chromosomal DNA. • They usually occur in bacteria, sometimes in eukaryotic organisms • Their size varies from 1 to 250 kilo base pairs (kbp). There are from one copy, for large plasmids, to hundreds of copies of the same plasmid present in a single cell. Ulf Schmitz, Introduction to genomics and proteomics I 18
  • Prokaryotic model organisms www. .uni-rostock.de E.coli (Escherichia coli) Methanococcus jannaschii (archaeon) Mycoplasma genitalium (simplest organism known) Ulf Schmitz, Introduction to genomics and proteomics I 19
  • www. Genomics .uni-rostock.de • DNA of higher organisms is organized into chromosomes (human – 23 chromosome pairs) • not all DNA codes for proteins • on the other hand some genes exist in multiple copies • that’s why from the genome size you can’t easily estimate the amount of protein sequence information Ulf Schmitz, Introduction to genomics and proteomics I 20
  • www. Genomes of eukaryotes .uni-rostock.de • majority of the DNA is in the nucleus, separated into bundles (chromosomes) – small amounts of DNA appear in organelles (mitochondria and chloroplasts) • within single chromosomes gene families are common – some family members are paralogues (related) • they have duplicated within the same genome • often diverged to provide separate functions in descendants (Nachkommen) • e.g. human α and β globin – orthologues genes • are homologues in different species • often perform the same function • e.g. human and horse myoglobin – pseudogenes • lost their function • e.g. human globin gene cluster pseudogene Ulf Schmitz, Introduction to genomics and proteomics I 21
  • Eukaryotic model organisms www. .uni-rostock.de • Saccharomyces cerevisiae (baker’s yeast) • Caenorhabditis elegans (C.elegans) • Drosophila melanogaster (fruit fly) • Arabidopsis thaliana (flower) • Homo sapiens (human) Ulf Schmitz, Introduction to genomics and proteomics I 22
  • The human genome • • • • • • • • • • • • • • www. .uni-rostock.de ~3.2 x 109 bp (thirty time larger than C.elegans or D.melongaster) coding sequences form only 5% of the human genome Repeat sequences over 50% Only ~32.000 genes Human genome is distributed over 22 chromosome pairs plus X and Y chromosomes Exons of protein-coding genes are relatively small compared to other known eukaryotic genomes Introns are relatively long Protein-coding genes span long stretches of DNA (dystrophin, coding a 3.685 amino acid protein, is >2.4Mbp long) Average gene length: ~ 8,000 bp Average of 5-6 exons/gene Average exon length: ~200 bp Average intron length: ~2,000 bp ~8% genes have a single exon Some exons can be as small as 1 or 3 bp. Ulf Schmitz, Introduction to genomics and proteomics I 23
  • www. The human genome .uni-rostock.de Top categories in a function classification: Function Number Nucleic acid binding DNA binding DNA repair protein DNA replication factor Transcription factor RNA binding Structural protein of ribosome Translation factor % 2207 1656 45 7 986 380 137 44 14.0 10.5 0.2 0.0 6.2 2.4 0.8 0.2 6 0.0 75 0.4 154 0.9 85 0.5 Actin binding 129 0.8 Defense/immunity protein 603 3.8 3242 457 403 839 295 20.6 2.9 2.5 5.3 1.8 3 0.0 Transcription factor binding Cell Cycle regulator Chaperone Motor Enzyme Peptidase Endopeptidase Protein kinase Protein phosphatase Enzyme activator Function Apoptosis inhibitor Number % 132 0.8 1790 1318 1202 489 71 11.4 8.4 7.6 3.1 0.0 7 0.0 Cell adhesion 189 1.2 Structural protein Cytoskeletal structural protein 714 145 4.5 0.9 Transporter Ion channel Neurotransmitter transporter 682 269 19 4.3 1.7 0.1 1536 33 50 9.7 0.2 0.3 5 0.0 4813 30.6 15683 100.0 Signal transduction Receptor Transmembrane receptor G-protein link receptor Olfactory receptor Storage protein Ligand binding or carrier Electron transfer Cytochrome P450 Tumor suppressor Unclassified Total Ulf Schmitz, Introduction to genomics and proteomics I 24
  • www. The human genome • .uni-rostock.de Repeated sequences comprise over 50% of the genome: – Transposable elements, or interspersed repeats include LINEs and SINEs (almost 50%) – Retroposed pseudogenes – Simple ‘stutters’ - repeats of short oligomers (minisatellites and microsatellites) – Segment duplication, of blocks of ~10 - 300kb – Blocks of tandem repeats, including gene families Element Size (bp) Short Interspersed Nuclear Elements (SINEs) 100-300 Long Interspersed Nuclear Elements (LINEs) Copy number Fraction of genome % 1.500.000 13 6000-8000 850.000 21 Long Terminal Repeats 15.000 -110.000 450.000 8 DNA Transposon fossils 80-3000 300.000 3 Ulf Schmitz, Introduction to genomics and proteomics I 25
  • The human genome www. .uni-rostock.de • All people are different, but the DNA of different people only varies for 0.2% or less. • So, only up to 2 letters in 1000 are expected to be different. • Evidence in current genomics studies (Single Nucleotide Polymorphisms or SNPs) imply that on average only 1 letter out of 1400 is different between individuals. • means that 2 to 3 million letters would differ between individuals. Ulf Schmitz, Introduction to genomics and proteomics I 26
  • www. Functional Genomics .uni-rostock.de From gene to function Genome Expressome Proteome TERTIARY STRUCTURE (fold) TERTIARY STRUCTURE (fold) Metabolome Ulf Schmitz, Introduction to genomics and proteomics I 27
  • DNA makes RNA makes Protein: www. .uni-rostock.de Expression data • More copies of mRNA for a gene leads to more protein • mRNA can now be measured for all the genes in a cell at ones through microarray technology • Can have 60,000 spots (genes) on a single gene chip • Color change gives intensity of gene expression (over- or under-expression) Ulf Schmitz, Introduction to genomics and proteomics I 28
  • www. Ulf Schmitz, Introduction to genomics and proteomics I .uni-rostock.de 29
  • Genes and regulatory regions www. .uni-rostock.de regulatory mechanisms organize the expression of genes – genes may be turned on or off in response to concentrations of nutrients or to stress – control regions often lie near the segments coding for proteins – they can serve as binding sites for molecules that transcribe the DNA – or they bind regulatory molecules that can block transcription Ulf Schmitz, Introduction to genomics and proteomics I 30
  • Expression data Ulf Schmitz, Introduction to genomics and proteomics I www. .uni-rostock.de 31
  • Outlook – coming lecture www. .uni-rostock.de Proteomics – Proteins – post-translational modification – Key technologies • Maps of hereditary information • SNPs (Single nucleotide polymorphisms) • Genetic diseases Ulf Schmitz, Introduction to genomics and proteomics I 32
  • www. .uni-rostock.de Thanks for your attention! Ulf Schmitz, Introduction to genomics and proteomics I 33
  • www. .uni-rostock.de Bioinformatics Introduction to genomics and proteomics II ulf.schmitz@informatik.uni-rostock.de Bioinformatics and Systems Biology Group www.sbi.informatik.uni-rostock.de Ulf Schmitz, Introduction to genomics and proteomics II 1
  • www. Outline .uni-rostock.de 1. Proteomics • • • • Motivation Post -Translational Modifications Key technologies Data explosion 2. Maps of hereditary information 3. Single nucleotide polymorphisms Ulf Schmitz, Introduction to genomics and proteomics II 2
  • www. Protomics .uni-rostock.de Proteomics: • is the large-scale study of proteins, particularly their structures and functions • This term was coined to make an analogy with genomics, and is often viewed as the "next step", • but proteomics is much more complicated than genomics. • Most importantly, while the genome is a rather constant entity, the proteome is constantly changing through its biochemical interactions with the genome. • One organism will have radically different protein expression in different parts of its body and in different stages of its life cycle. Proteome: The entirety of proteins in existence in an organism are referred to as the proteome. Ulf Schmitz, Introduction to genomics and proteomics II 3
  • www. Proteomics .uni-rostock.de If the genome is a list of the instruments in an orchestra, the proteome is the orchestra playing a symphony. R.Simpson Ulf Schmitz, Introduction to genomics and proteomics II 4
  • www. Proteomics • • .uni-rostock.de Describing all 3D structures of proteins in the cell is called Structural Genomics Finding out what these proteins do is called Functional Genomics DNA Microarray GENOME Genetic Screens PROTEOME Protein – Protein Interactions Protein – Ligand Interactions Structure Ulf Schmitz, Introduction to genomics and proteomics II 5
  • www. Proteomics .uni-rostock.de Motivation: • What kind of data would we like to measure? • What mature experimental techniques exist to determine them? • The basic goal is a spatio-temporal description of the deployment of proteins in the organism. Ulf Schmitz, Introduction to genomics and proteomics II 6
  • www. Proteomics .uni-rostock.de Things to consider: • the rates of synthesis of different proteins vary among different tissues and different cell types and states of activity • methods are available for efficient analysis of transcription patterns of multiple genes • because proteins ‘turn over’ at different rates, it is also necessary to measure proteins directly • the distribution of expressed protein levels is a kinetic balance between rates of protein synthesis and degradation Ulf Schmitz, Introduction to genomics and proteomics II 7
  • www. Ulf Schmitz, Introduction to genomics and proteomics II .uni-rostock.de 8
  • Why do Proteomics? • www. .uni-rostock.de are there differences between amino acid sequences determined directly from proteins and those determined by translation from DNA? – pattern recognition programs addressing this questions have following errors: • • • • • a genuine protein sequence may be missed entirely an incomplete protein may be reported a gene may be incorrectly spliced genes for different proteins may overlap genes may be assembled from exons in different ways in different tissues – often, molecules must be modified to make a mature protein that differs significantly from the one suggested by translation • in many cases the missing post-translational- modifications are quite important and have functional significance • post-transitional modifications include addition of ligands, glycosylation, methylation, excision of peptides, etc. – in some cases mRNA is edited before translation, creating changes in the amino acid sequence that are not inferrable from the genes • a protein inferred from a genome sequence is a hypothetical object until an experiment verifies its existence Ulf Schmitz, Introduction to genomics and proteomics II 9
  • Post-translational modification www. .uni-rostock.de • a protein is a polypeptide chain composed of 20 possible amino acids • there are far fewer genes that code for proteins in the human genome than there are proteins in the human proteome (~33,000 genes vs ~200,000 proteins). • each gene encodes as many as six to eight different proteins – due to post-translational modifications such as phosphorylation, glycosylation or cleavage (Spaltung) • posttranslational modification extends the range of possible functions a protein can have – changes may alter the hydrophobicity of a protein and thus determine if the modified protein is cytosolic or membrane-bound – modifications like phosphorylation are part of common mechanisms for controlling the behavior of a protein, for instance, activating or inactivating an enzyme. Ulf Schmitz, Introduction to genomics and proteomics II 10
  • Post-translational modification www. .uni-rostock.de Phosphorylation • • • • phosphorylation is the addition of a phosphate (PO4) group to a protein or a small molecule (usual to serine, tyrosine, threonine or histidine) In eukaryotes, protein phosphorylation is probably the most important regulatory event Many enzymes and receptors are switched "on" or "off" by phosphorylation and dephosphorylation Phosphorylation is catalyzed by various specific protein kinases, whereas phosphatases dephosphorylate. Acetylation • Is the addition of an acetyl group, usually at the N-terminus of the protein Farnesylation • farnesylation, the addition of a farnesyl group Glycosylation • the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein Ulf Schmitz, Introduction to genomics and proteomics II 11
  • www. Proteomics Ulf Schmitz, Introduction to genomics and proteomics II .uni-rostock.de 12
  • Key technologies for proteomics www. .uni-rostock.de 1. 1-D electrophoresis and 2-D electrophoresis • are for the separation and visualization of proteins. 2. mass spectrometry, x-ray crystallography, and NMR (Nuclear magnetic resonance ) • are used to identify and characterize proteins 3. chromatography techniques especially affinity chromatography • are used to characterize protein-protein interactions. 4. Protein expression systems like the yeast twohybrid and FRET (fluorescence resonance energy transfer) • can also be used to characterize protein-protein interactions. Ulf Schmitz, Introduction to genomics and proteomics II 13
  • Key technologies for proteomics www. .uni-rostock.de High-resolution two-dimensional polyacrylamide gel electrophoresis (2D PAGE) shows the pattern of protein content in a sample. Reference map of lympphoblastoid cell linePRI, soluble proteins. • 110 µg of proteins loaded • Strip 17cm pH gradient 4-7, SDS PAGE gels 20 x 25 cm, 8-18.5% T. • Staining by silver nitrate method (Rabilloud et al.,) • Identification by mass spectrometry. The pinks labels on the spots indicate the ID in Swiss-prot database browse the SWISS-2DPAGE database for more 2d PAGE images Ulf Schmitz, Introduction to genomics and proteomics II 14
  • www. Proteomics .uni-rostock.de X-ray crystallography is a means to determine the detailed molecular structure of a protein, nucleic acid or small molecule. With a crystal structure we can explain the mechanism of an enzyme, the binding of an inhibitor, the packing of protein domains, the tertiary structure of a nucleic acid molecule etc.. Typically, a sample is purified to homogeneity, crystallized, subjected to an Xray beam and diffraction data are collected. Ulf Schmitz, Introduction to genomics and proteomics II 15
  • High-throughput Biological Data www. .uni-rostock.de • Enormous amounts of biological data are being generated by high-throughput capabilities; even more are coming – – – – – – genomic sequences gene expression data (microarrays) mass spec. data protein-protein interaction (chromatography) protein structures (x-ray christallography) ...... Ulf Schmitz, Introduction to genomics and proteomics II 16
  • Protein structural data explosion www. .uni-rostock.de Protein Data Bank (PDB): 33.367 Structures (1 November 2005) 28.522 x-ray crystallography, 4.845 NMR Ulf Schmitz, Introduction to genomics and proteomics II 17
  • Maps of hereditary information www. .uni-rostock.de Following maps are used to find out how hereditary information is stored, passed on, and implemented. 1. Linkage maps of genes mini- / microsatellites 2. Banding patterns of chromosomes physical objects with visible landmarks called banding patterns 3. DNA sequences Contig maps (contigous clone maps) Sequence tagged site (STS) SNPs (Single nucloetide polymorphisms) Ulf Schmitz, Introduction to genomics and proteomics II 18
  • www. .uni-rostock.de Linkage map Ulf Schmitz, Introduction to genomics and proteomics II 19
  • Maps of hereditary information www. .uni-rostock.de Variable number tandem repeats (VNTRs, also minisatellites) • regions, 8-80bp long, repeated a variable number of times • the distribution and the size of repeats is the marker • inheritance of VNTRs can be followed in a family and mapped to a pathological phenotype • first genetic data used for personal identification – Genetic fingerprints; in paternity and in criminal cases Short tandem repeat polymorphism (STRPs, also microsatellites) • Regions of 2-7bp, repeated many times – Usually 10-30 consecutive copies Ulf Schmitz, Introduction to genomics and proteomics II 20
  • www. .uni-rostock.de centromere 3bp CGTCGTCGTCGTCGTCGTCGTCGT... GCAGCAGCAGCAGCAGCAGCAGCA... Ulf Schmitz, Introduction to genomics and proteomics II 21
  • Maps of hereditary information www. .uni-rostock.de Banding patterns of chromosomes Ulf Schmitz, Introduction to genomics and proteomics II 22
  • Maps of hereditary information www. .uni-rostock.de Banding patterns of chromosomes petite – arm centromere queue - arm Ulf Schmitz, Introduction to genomics and proteomics II 23
  • Maps of hereditary information www. .uni-rostock.de Contig map (also contiguous clone map) • • • Series of overlapping DNA clones of known order along a chromosome from an organism of interest, stored in yeast or bacterial cells as YACs (Yeast Artificial Chromosomes) or BACs (Bacterial Artificial Chromosomes) A contig map produces a fine mapping (high resolution) of a genome YAC can contain up to 106bp, a BAC about 250.000bp Sequence tagged site (STS) • • Short, sequenced region of DNA, 200-600bp long, that appears in a unique location in the genome One type arises from an EST (expressed sequence tag), a piece of cDNA Ulf Schmitz, Introduction to genomics and proteomics II 24
  • Maps of hereditary information www. .uni-rostock.de Imagine we know that a disease results from a specific defective protein: 1. if we know the protein involved, we can pursue rational approaches to therapy 2. if we know the gene involved, we can devise tests to identify sufferers or carriers 3. wereas the knowledge of the chromosomal location of the gene is unnecessary in many cases for either therapy or detection; • it is required only for identifying the gene, providing a bridge between the patterns of inheritance and the DNA sequence Ulf Schmitz, Introduction to genomics and proteomics II 25
  • Single nucleotide polymorphisms (SNPs) • • • www. .uni-rostock.de SNP (pronounced ‘snip’) is a genetic variation between individuals single base pairs that can be substituted, deleted or inserted SNPs are distributed throughout the genome – average every 2000bp • • provide markers for mapping genes not all SNPs are linked to diseases Ulf Schmitz, Introduction to genomics and proteomics II 26
  • Single nucleotide polymorphisms (SNPs) www. .uni-rostock.de • nonsense mutations: – codes for a stop, which can truncate the protein • missense mutations: – codes for a different amino acid • silent mutations: – codes for the same amino acid, so has no effect Ulf Schmitz, Introduction to genomics and proteomics II 27
  • Outlook – coming lecture www. .uni-rostock.de • Bioinformatics Information Resources And Networks – EMBnet – European Molecular Biology Network • DBs and Tools – NCBI – National Center For Biotechnology Information • DBs and Tools – – – – – – Nucleic Acid Sequence Databases Protein Information Resources Metabolic Databases Mapping Databases Databases concerning Mutations Literature Databases Ulf Schmitz, Introduction to genomics and proteomics II 28
  • www. .uni-rostock.de Thanks for your attention! Ulf Schmitz, Introduction to genomics and proteomics II 29