Peptide mass fingerprinting - wikipedia Peptide mass fingerprinting Jump to : navigation , search A typical workflow of a peptide mass fingerprinting experiment . Peptide mass fingerprinting ( PMF ) ( also known as protein fingerprinting ) is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides , whose absolute masses can be accurately measured with a mass spectrometer such as MALDI - TOF or ESI - TOF . The method was developed in 1993 by several groups independently . The peptide masses are compared to either a database containing known protein sequences or even the genome . This is achieved by using computer programs that translate the known genome of the organism into proteins , then theoretically cut the proteins into peptides , and calculate the absolute masses of the peptides from each protein . They then compare the masses of the peptides of the unknown protein to the theoretical peptide masses of each protein encoded in the genome . The results are statistically analyzed to find the best match . The advantage of this method is that only the masses of the peptides have to be known . Time - consuming de novo peptide sequencing is then unnecessary . A disadvantage is that the protein sequence has to be present in the database of interest . Additionally most PMF algorithms assume that the peptides come from a single protein . The presence of a mixture can significantly complicate the analysis and potentially compromise the results . Typical for the PMF based protein identification is the requirement for an isolated protein . Mixtures exceeding a number of 2 - 3 proteins typically require the additional use of MS / MS based protein identification to achieve sufficient specificity of identification ( 6 ) . Therefore , the typical PMF samples are isolated proteins from two - dimensional gel electrophoresis ( 2D gels ) or isolated SDS - PAGE bands . Additional analyses by MS / MS can either be direct , e.g. , MALDI - TOF / TOF analysis or downstream nanoLC - ESI - MS / MS analysis of gel spot eluates . Contents ( hide ) 1 Origins 2 Sample preparation 3 Mass spectrometric analysis 4 Computational analysis 5 See also 6 References 7 External links Origins ( edit ) Due to the long , tedious process of analyzing proteins , peptide mass fingerprinting was developed . Edman degradation was used in protein analysis , and it required almost an hour to analyze one amino acid residue . SDS - PAGE was also used to separate proteins in very complex mixtures , which also employed methods of electroblotting and staining . Then , bands would be extracted from the gel and sequenced , automatically . A recurring problem in the process was that interfering proteins would also purify with the protein of interest . The sequences of these interfering proteins were compiled into what came to known as the Dayhoff database . Ultimately , having the sequences of these known protein contaminants in databases decreased instrument time and expenses involved in protein analysis . Sample preparation ( edit ) Protein samples can be derived from SDS - PAGE or reversed phase HPLC , and are then subject to some chemical modifications . Disulfide bridges in proteins are reduced and cysteine amino acids are carbamidomethylated chemically or acrylamidated during the gel electrophoresis . Then the proteins are cut into several fragments using proteolytic enzymes such as trypsin , chymotrypsin or Glu - C. A typical sample : protease ratio is 50 : 1 . The proteolysis is typically carried out overnight and the resulting peptides are extracted with acetonitrile and dried under vacuum . The peptides are then dissolved in a small amount of distilled water or further concentrated and purified and are ready for mass spectrometric analysis . Mass spectrometric analysis ( edit ) The digested protein can be analyzed with different types of mass spectrometers such as ESI - TOF or MALDI - TOF . MALDI - TOF is often the preferred instrument because it allows a high sample throughput and several proteins can be analyzed in a single experiment , if complemented by MS / MS analysis . LC / ESI - MS and CE / ESI - MS are also great techniques for peptide mass fingerprinting . A small fraction of the peptide ( usually 1 microliter or less ) is pipetted onto a MALDI target and a chemical called a matrix is added to the peptide mix . Common matrices are Sinapinic acid , Alpha - Cyano - 4 - hydroxycinnamic acid , and 2 , 3 - Dihydroxybenzoic acid . The matrix molecules are required for the desorption of the peptide molecules . Matrix and peptide molecules co-crystallize on the MALDI target and are ready to be analyzed . There is one predominantly MALDI - MS sample preparation technique , namely dried droplet technique . The target is inserted into the vacuum chamber of the mass spectrometer and the desorption and ionisation of the polypeptide fragments is initiated by a pulsed laser beam which transfers high amounts of energy into the matrix molecules . The energy transfer is sufficient to promote the ionisation and transition of matrix molecules and peptides from the solid phase into the gas phase . The ions are accelerated in the electric field of the mass spectrometer and fly towards an ion detector where their arrival is detected as an electric signal . Their mass - to - charge ratio is proportional to their time of flight ( TOF ) in the drift tube and can be calculated accordingly . Coupling ESI with capillary LC can separate peptides from protein digests , while obtaining their molecular masses at the same time . Capillary electrophoresis coupled with ESI - MS is another technique ; however , it works best when analyzing small amounts of proteins . Computational analysis ( edit ) The mass spectrometric analysis produces a list of molecular weights of the fragments which is often called a peak list . The peptide masses are compared to protein databases such as Swissprot , which contain protein sequence information . Software performs in silico digests on proteins in the database with the same enzyme ( e.g. trypsin ) used in the chemical cleavage reaction . The mass of these peptide fragments is then calculated and compared to the peak list of measured peptide masses . The results are statistically analyzed and possible matches are returned in a results table . See also ( edit ) Protein mass spectrometry Shotgun proteomics Top - down proteomics Bottom - up proteomics References ( edit ) Jump up ^ Clauser KR , Baker P , Burlingame AL ( 1999 ) . `` Role of accurate mass measurement ( + / - 10 ppm ) in protein identification strategies employing MS or MS / MS and database searching '' . Anal . Chem. 71 ( 14 ) : 2871 -- 82 . doi : 10.1021 / ac9810516 . PMID 10424174 . Jump up ^ Pappin DJ , Hojrup P , Bleasby AJ ( 1993 ) . `` Rapid identification of proteins by peptide - mass fingerprinting '' . Curr. Biol. 3 ( 6 ) : 327 -- 32 . doi : 10.1016 / 0960 - 9822 ( 93 ) 90195 - T. PMID 15335725 . Jump up ^ Henzel WJ , Billeci TM , Stults JT , Wong SC , Grimley C , Watanabe C ( 1993 ) . `` Identifying proteins from two - dimensional gels by molecular mass searching of peptide fragments in protein sequence databases '' . Proc. Natl. Acad. Sci. U.S.A. 90 ( 11 ) : 5011 -- 5 . Bibcode : 1993PNAS ... 90.5011 H . doi : 10.1073 / pnas. 90.11. 5011 . PMC 46643 . PMID 8506346 . Jump up ^ Mann M , Højrup P , Roepstorff P ( 1993 ) . `` Use of mass spectrometric molecular weight information to identify proteins in sequence databases '' . Biol. Mass Spectrom. 22 ( 6 ) : 338 -- 45 . doi : 10.1002 / bms. 1200220605 . PMID 8329463 . Jump up ^ James P , Quadroni M , Carafoli E , Gonnet G ( 1993 ) . `` Protein identification by mass profile fingerprinting '' . Biochem. Biophys . Res . Commun. 195 ( 1 ) : 58 -- 64 . doi : 10.1006 / bbrc. 1993.2009 . PMID 8363627 . Jump up ^ Yates JR , Speicher S , Griffin PR , Hunkapiller T ( 1993 ) . `` Peptide mass maps : a highly informative approach to protein identification '' . Anal . Biochem. 214 ( 2 ) : 397 -- 408 . doi : 10.1006 / abio. 1993.1514 . PMID 8109726 . ^ Jump up to : Shevchenko A , Jensen ON , Podtelejnikov AV , Sagliocco F , Wilm M , Vorm O , Mortensen P , Shevchenko A , Boucherie H , Mann M ( 1996 ) . `` Linking genome and proteome by mass spectrometry : large - scale identification of yeast proteins from two dimensional gels '' . Proc. Natl. Acad. Sci. U.S.A. 93 ( 25 ) : 14440 -- 5 . Bibcode : 1996PNAS ... 9314440S . doi : 10.1073 / pnas. 93.25. 14440 . PMC 26151 . PMID 8962070 . Jump up ^ Wang W , Sun J , Nimtz M , Deckwer WD , Zeng AP ( 2003 ) . `` Protein identification from two - dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences '' . Proteome Science . 1 ( 1 ) : 6 . doi : 10.1186 / 1477 - 5956 - 1 - 6 . PMC 317362 . PMID 14653859 . Jump up ^ Henzel , William J. ; Watanabe , Colin ; Stults , John T. ( 2003 - 09 - 01 ) . `` Protein identification : The origins of peptide mass fingerprinting '' . Journal of the American Society for Mass Spectrometry. 14 ( 9 ) : 931 -- 942 . doi : 10.1016 / S1044 - 0305 ( 03 ) 00214 - 9 . ISSN 1044 - 0305 . Jump up ^ Matsudaira , P. ( 1987 - 07 - 25 ) . `` Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes '' . The Journal of Biological Chemistry . 262 ( 21 ) : 10035 -- 10038 . ISSN 0021 - 9258 . PMID 3611052 . Jump up ^ BC Orcutt ; DG George ; Dayhoff , and M.O. ( 1983 ) . `` Protein and Nucleic Acid Sequence Database Systems '' . Annual Review of Biophysics and Bioengineering. 12 ( 1 ) : 419 -- 441 . doi : 10.1146 / annurev. bb. 12.060183. 002223 . PMID 6347043 . Jump up ^ Moore , R.E. ; Licklider , L. ; Schumann , D. ; Lee , T.D. ( 1998 - 12 - 01 ) . `` A microscale electrospray interface incorporating a monolithic , poly ( styrene - divinylbenzene ) support for on - line liquid chromatography / tandem mass spectrometry analysis of peptides and proteins '' . Analytical Chemistry . 70 ( 23 ) : 4879 -- 4884 . ISSN 0003 - 2700 . PMID 9852776 . ^ Jump up to : Whitmore , Colin D. ; Gennaro , Lynn A. ( 2012 - 06 - 01 ) . `` Capillary electrophoresis - mass spectrometry methods for tryptic peptide mapping of therapeutic antibodies '' . Electrophoresis. 33 ( 11 ) : 1550 -- 1556 . doi : 10.1002 / elps. 201200066 . ISSN 1522 - 2683 . Jump up ^ Thiede , Bernd ( 2005 ) . `` Peptide mass fingerprinting '' . Methods. 35 ( 3 ) : 237 -- 247 . doi : 10.1016 / j. ymeth. 2004.08. 015 -- via Elsevier . Jump up ^ Dass , Chhabil . Fundamentals of Contemporary Mass Spectrometry Wiley Online Books . doi : 10.1002 / 0470118490 . External links ( edit ) Library resources about Peptide mass fingerprinting Resources in your library Resources in other libraries ( hide ) Proteins : key methods of study Experimental Protein purification Green fluorescent protein Western blot Protein immunostaining Protein sequencing Gel electrophoresis / Protein electrophoresis Protein immunoprecipitation Peptide mass fingerprinting / Protein mass spectrometry Dual - polarization interferometry Microscale thermophoresis Chromatin immunoprecipitation Surface plasmon resonance Isothermal titration calorimetry X-ray crystallography Protein NMR Cryo - electron microscopy Freeze - fracture electron microscopy Bioinformatics Protein structure prediction Protein -- protein docking Protein structural alignment Protein ontology Protein -- protein interaction prediction Assay Enzyme assay Protein assay Secretion assay Display techniques Bacterial display mRNA display Phage display Ribosome display Yeast display Super-resolution microscopy Photoactivated localization microscopy Vertico SMI Retrieved from `` https://en.wikipedia.org/w/index.php?title=Peptide_mass_fingerprinting&oldid=838448080 '' Categories : Protein methods Laboratory techniques Mass spectrometry Talk Contents About Wikipedia Deutsch Español 日本 語 Српски / srpski Edit links This page was last edited on 27 April 2018 , at 02 : 23 . About Wikipedia 
protein identification the origins of peptide mass fingerprinting
Peptide mass fingerprinting ( PMF ) ( also known as protein fingerprinting ) is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides , whose absolute masses can be accurately measured with a mass spectrometer such as MALDI - TOF or ESI - TOF . The method was developed in 1993 by several groups independently . The peptide masses are compared to either a database containing known protein sequences or even the genome . This is achieved by using computer programs that translate the known genome of the organism into proteins , then theoretically cut the proteins into peptides , and calculate the absolute masses of the peptides from each protein . They then compare the masses of the peptides of the unknown protein to the theoretical peptide masses of each protein encoded in the genome . The results are statistically analyzed to find the best match . The advantage of this method is that only the masses of the peptides have to be known . Time - consuming de novo peptide sequencing is then unnecessary . A disadvantage is that the protein sequence has to be present in the database of interest . Additionally most PMF algorithms assume that the peptides come from a single protein . The presence of a mixture can significantly complicate the analysis and potentially compromise the results . Typical for the PMF based protein identification is the requirement for an isolated protein . Mixtures exceeding a number of 2 - 3 proteins typically require the additional use of MS / MS based protein identification to achieve sufficient specificity of identification ( 6 ) . Therefore , the typical PMF samples are isolated proteins from two - dimensional gel electrophoresis ( 2D gels ) or isolated SDS - PAGE bands . Additional analyses by MS / MS can either be direct , e.g. , MALDI - TOF / TOF analysis or downstream nanoLC - ESI - MS / MS analysis of gel spot eluates . Due to the long , tedious process of analyzing proteins , peptide mass fingerprinting was developed . Edman degradation was used in protein analysis , and it required almost an hour to analyze one amino acid residue . SDS - PAGE was also used to separate proteins in very complex mixtures , which also employed methods of electroblotting and staining . Then , bands would be extracted from the gel and sequenced , automatically . A recurring problem in the process was that interfering proteins would also purify with the protein of interest . The sequences of these interfering proteins were compiled into what came to known as the Dayhoff database . Ultimately , having the sequences of these known protein contaminants in databases decreased instrument time and expenses involved in protein analysis . Protein samples can be derived from SDS - PAGE or reversed phase HPLC , and are then subject to some chemical modifications . Disulfide bridges in proteins are reduced and cysteine amino acids are carbamidomethylated chemically or acrylamidated during the gel electrophoresis . Then the proteins are cut into several fragments using proteolytic enzymes such as trypsin , chymotrypsin or Glu - C. A typical sample : protease ratio is 50 : 1 . The proteolysis is typically carried out overnight and the resulting peptides are extracted with acetonitrile and dried under vacuum . The peptides are then dissolved in a small amount of distilled water or further concentrated and purified and are ready for mass spectrometric analysis . The digested protein can be analyzed with different types of mass spectrometers such as ESI - TOF or MALDI - TOF . MALDI - TOF is often the preferred instrument because it allows a high sample throughput and several proteins can be analyzed in a single experiment , if complemented by MS / MS analysis . LC / ESI - MS and CE / ESI - MS are also great techniques for peptide mass fingerprinting . A small fraction of the peptide ( usually 1 microliter or less ) is pipetted onto a MALDI target and a chemical called a matrix is added to the peptide mix . Common matrices are Sinapinic acid , Alpha - Cyano - 4 - hydroxycinnamic acid , and 2 , 3 - Dihydroxybenzoic acid . The matrix molecules are required for the desorption of the peptide molecules . Matrix and peptide molecules co-crystallize on the MALDI target and are ready to be analyzed . There is one predominantly MALDI - MS sample preparation technique , namely dried droplet technique . The target is inserted into the vacuum chamber of the mass spectrometer and the desorption and ionisation of the polypeptide fragments is initiated by a pulsed laser beam which transfers high amounts of energy into the matrix molecules . The energy transfer is sufficient to promote the ionisation and transition of matrix molecules and peptides from the solid phase into the gas phase . The ions are accelerated in the electric field of the mass spectrometer and fly towards an ion detector where their arrival is detected as an electric signal . Their mass - to - charge ratio is proportional to their time of flight ( TOF ) in the drift tube and can be calculated accordingly . Coupling ESI with capillary LC can separate peptides from protein digests , while obtaining their molecular masses at the same time . Capillary electrophoresis coupled with ESI - MS is another technique ; however , it works best when analyzing small amounts of proteins . The mass spectrometric analysis produces a list of molecular weights of the fragments which is often called a peak list . The peptide masses are compared to protein databases such as Swissprot , which contain protein sequence information . Software performs in silico digests on proteins in the database with the same enzyme ( e.g. trypsin ) used in the chemical cleavage reaction . The mass of these peptide fragments is then calculated and compared to the peak list of measured peptide masses . The results are statistically analyzed and possible matches are returned in a results table .