Proteomics made more accessible

MS‐based proteomics is a bioinformatic‐intensive field. Additionally, the instruments and instrument‐related and analytic software are expensive. Some free Internet‐based proteomics tools have gained wide usage, but there have not been any single bioinformatic framework that in an easy and intuitive way guided the user through the whole process from analyses to submission. Together, these factors may have limited the expansion of proteomics analyses, and also the secondary use (reanalyses) of proteomic data. Vaudel et al. (Proteomics 2014, 14, 1001–1005) are now describing their Compomics framework that guides the user through all the main steps, from the database generation, via the analyses and validation, and through the submission process to PRIDE, a proteomic data bank. Vaudel et al. partly base the framework on tools that they have developed themselves, and partly they are integrating other freeware tools into the workflow. One of the most interesting aspects with the Compomics framework is the possibility of extending MS‐based proteomics outside the MS laboratory itself. With the Compomics framework, any laboratory can handle large amounts of proteomic data, thereby facilitating collaboration and in‐depth data analyses. The described software also opens the potential for any laboratory to reanalyze data deposited in PRIDE.     

Label‐free analysis of human cerebrospinal fluid addressing various normalization strategies and revealing protein groups affected by multiple sclerosis

The aims of the study were to: (i) identify differentially regulated proteins in cerebrospinal fluid (CSF) between multiple sclerosis (MS) patients and non‐MS controls; (ii) examine the effect of matching the CSF samples on either total protein amount or volume, and compare four protein normalization strategies for CSF protein quantification. CSF from MS patients (n = 37) and controls (n = 64), consisting of other noninflammatory neurological diseases (n = 50) and non neurological spinal anesthetic subjects (n = 14), were analyzed using label‐free proteomics, quantifying almost 800 proteins. In total, 122 proteins were significantly regulated (p < 0.05), where 77 proteins had p‐value <0.01 or AUC value >0.75. Hierarchical clustering indicated that there were two main groups of MS patients, those with increased levels of inflammatory response proteins and decreased levels of proteins involved in neuronal tissue development (n = 30), and those with normal protein levels for both of these protein groups (n = 7). The main subgroup of controls clustering with the MS patients showing increased inflammation and decreased neuronal tissue development were patients suffering from chronic fatigue. Our data indicate that the preferable way to quantify proteins in CSF is to first match the samples on total protein amount and then normalize the data based on the median intensities, preferably from the CNS‐enriched proteins.     

High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry

As the number of fully sequenced animal genomes and the performance of advanced mass spectrometry-based proteomics techniques are continuously improving, there is now a great opportunity to increase the knowledge of various animal proteomes. This research area is further stimulated by a growing interest from veterinary medicine and the pharmaceutical industry. Cerebrospinal fluid (CSF) is a good source for better understanding of diseases related to the central nervous system, both in humans and other animals.In this study, four high-abundant protein depletion columns, developed for human or rat serum, were evaluated for dog CSF. For the analysis, a shotgun proteomics approach, based on nanoLC-LTQ Orbitrap MS/MS, was applied. All the selected approaches were shown to deplete dog CSF with different success. It was demonstrated that the columns significantly improved the coverage of the detected dog CSF proteome. An antibody-based column showed the best performance, in terms of efficiency, repeatability and the number of proteins detected in the sample. In total 983 proteins were detected. Of those, 801 proteins were stated as uncharacterized in the UniProt database. To the best of our knowledge, this is the so far largest number of proteins reported for dog CSF in one single study.     

In this issue: Proteomics 20/2008

In this issue of Proteomics you will find the following highlighted articles: Comprehensively confirmed annotation of short ORFs During the California gold rush, prospectors did not look only for large nuggets to pave their road to riches – they also collected gold dust. The proteomic equivalent of dust is the small ORF. It does not pay to put a lower limit on dust or ORF size because almost any size may be turned into “cash” or an important peptide. Wei et al. demonstrate how a combination of conventional genomics and proteomics can find small expressed ORFs in noisy mixtures. Their test system is Shigella flexneri rRNA genes. First, cells were lysed, pre‐fractionated through 100 kDa and 10 kDa cutoff membranes and split into two aliquots, one treated with trypsin the other not. Both were analyzed by LC‐MS/MS and queried for ORFs >15 amino acids. Total labeled RNA probed a DNA array, and, after BLAST classification, revealed 36 new small proteins. Wei, C. et al., Proteomics 2008, 8, 4209–4213. The end of an era: Late stage proteome of cheese bacterium The bacterium Streptococcus thermophilus is a common ingredient in food, serving as a starter culture for a wide variety of dairy foods, yogurt in partic­ular, in co‐culture with Lactobacillus delbrueckii ssp. bulgaricus. It is closely related to several pathogenic species but lacks critical genes for pathogenicity. Instead, it is better optimized for lactose digestion than even a teen‐aged boy. Specific genes transport lactose into the cell where it is cleaved by a β‐galactosidase. Because milk is poor in amino acids, some strains of S. thermophilius bring their own protease to digest milk peptides and proteins. This work by Herve‐Jimenez et al. finds that in the latest stage of fermentation a number of critical changes occur: carbon sources are diversified, nitrogen uptake and biosynthesis increase, and there is a dramatic up‐regulation of the entire sulfur‐containing amino acid synthetic pathways. Stimulation by L. bulgaricus is a more complex effect. Herve‐Jimenez, L. et al., Proteomics 2008, 8, 4273–4286. 2000‐spot 2‐D DIGE gels yield interesting new CJD marker from CSF Creutzfeld‐Jakob disease (CJD) is a rare, fatal neurodegenerative disease, caused by prions. It is normally seen in either a sporadic (sCJD) or variant (vCJD) form. Differential diagnosis is difficult because the few tests available are weak in sensitivity or specificity, or have not been thoroughly validated. Brechlin et al. report here a much improved cerebrospinal fluid test based on 2‐D DIGE technology and improved sample preparation. Samples were concentrated 20‐fold, then depleted of serum albumin and immunoglobulins, and, finally, labeled with CyDyes. When separated on 24‐cm pH 4 – 7 IPG strips, then on 10 % PAGE‐SDS second dimension gels, >2000 spots could be resolved. After spot analysis for significant differences (volume, p‐value, variance) five spots remained and 4 of the 5 were identified by MALDI‐TOF‐MS. The unidentified spot showed the highest discrimination (p <0.001). Identification work for the fifth spot is continuing. Brechlin, P. et al., Proteomics 2008, 8, 4357–4366.     

Cover Picture: Proteomics 20/2008

In this issue of Proteomics you will find the following highlighted articles: Comprehensively confirmed annotation of short ORFs During the California gold rush, prospectors did not look only for large nuggets to pave their road to riches – they also collected gold dust. The proteomic equivalent of dust is the small ORF. It does not pay to put a lower limit on dust or ORF size because almost any size may be turned into “cash” or an important peptide. Wei et al. demonstrate how a combination of conventional genomics and proteomics can find small expressed ORFs in noisy mixtures. Their test system is Shigella flexneri rRNA genes. First, cells were lysed, pre‐fractionated through 100 kDa and 10 kDa cutoff membranes and split into two aliquots, one treated with trypsin the other not. Both were analyzed by LC‐MS/MS and queried for ORFs >15 amino acids. Total labeled RNA probed a DNA array, and, after BLAST classification, revealed 36 new small proteins. Wei, C. et al., Proteomics 2008, 8, 4209–4213. The end of an era: Late stage proteome of cheese bacterium The bacterium Streptococcus thermophilus is a common ingredient in food, serving as a starter culture for a wide variety of dairy foods, yogurt in partic­ular, in co‐culture with Lactobacillus delbrueckii ssp. bulgaricus. It is closely related to several pathogenic species but lacks critical genes for pathogenicity. Instead, it is better optimized for lactose digestion than even a teen‐aged boy. Specific genes transport lactose into the cell where it is cleaved by a β‐galactosidase. Because milk is poor in amino acids, some strains of S. thermophilius bring their own protease to digest milk peptides and proteins. This work by Herve‐Jimenez et al. finds that in the latest stage of fermentation a number of critical changes occur: carbon sources are diversified, nitrogen uptake and biosynthesis increase, and there is a dramatic up‐regulation of the entire sulfur‐containing amino acid synthetic pathways. Stimulation by L. bulgaricus is a more complex effect. Herve‐Jimenez, L. et al., Proteomics 2008, 8, 4273–4286. 2000‐spot 2‐D DIGE gels yield interesting new CJD marker from CSF Creutzfeld‐Jakob disease (CJD) is a rare, fatal neurodegenerative disease, caused by prions. It is normally seen in either a sporadic (sCJD) or variant (vCJD) form. Differential diagnosis is difficult because the few tests available are weak in sensitivity or specificity, or have not been thoroughly validated. Brechlin et al. report here a much improved cerebrospinal fluid test based on 2‐D DIGE technology and improved sample preparation. Samples were concentrated 20‐fold, then depleted of serum albumin and immunoglobulins, and, finally, labeled with CyDyes. When separated on 24‐cm pH 4 – 7 IPG strips, then on 10 % PAGE‐SDS second dimension gels, >2000 spots could be resolved. After spot analysis for significant differences (volume, p‐value, variance) five spots remained and 4 of the 5 were identified by MALDI‐TOF‐MS. The unidentified spot showed the highest discrimination (p <0.001). Identification work for the fifth spot is continuing. Brechlin, P. et al., Proteomics 2008, 8, 4357–4366.     

Cover Picture: Proteomics 5/2008

In this issue of Proteomics you will find the following highlighted articles: When is a stain not a stain? When it's dyeing! [Dumb proteomics joke!] This silly riddle is actually relat­ed to a recurrent question in proteomics: when is the best time to apply detection reagents to proteins for quantitative analysis? (a) pre‐electrophoresis labeling with DIGE/Cy‐type of covalent stains, or (b) post‐electrophoresis staining with silver, Sypro Ruby or Deep Purple? Karp et al. explore the question using a bacterial extract as a typical sample, DIGE Cy labels, and Deep Purple. It gets more complex when they have to deal with the “missingness” of spots: just because a spot doesn’t show up doesn’t mean it is not there, there just may not be enough to detect. Progenesis SameSpots software was used to analyze images for missing spots. In the end, DIGE gave better sensitivity as previously reported, and fewer missing spots. Deep Purple was more competitive when analyzed with SameSpots software. Karp, N. A. et al., Proteomics 2008, 8, 948–960. Your own best enemy? If there weren’t one maverick, black sheep, rebel, outlaw, eccentric, or rotten apple in most families, a lot of novels would never have been written. Mammalian immune systems seem to have the same structure – they mostly target enemies of the body but there always seem to be a few maverick antibodies that are targeted at their own body’s antigens. Servettaz et al. take up proteomic tools to identify the targets of the anti‐self antibodies expressed by apparently healthy individuals. Using umbilical cord endothelial cells as a source of antigens, the authors found 884 spots by ­2‐­DE, and 61 ± 25 of those were recognized by serum IgGs. All 12 sera tested recognized 11 antigens derived from 6 proteins. There were 3 cytoskeletal, 2 glycolytic, and 1 disulfide isomerase protein seen. These were confirmed by immunoblotting of 2‐D gels and identification by in‐gel tryptic digestion and MALDI‐TOF MS. Servettaz, A. et al., Proteomics 2008, 8, 1000–1008. Signature in scraps from kidney growth stages You can tell a lot about the quality of a new building, residential or commercial, by what doesn’t go into it. The scraps of lumber, pieces of masonry, lengths and varieties of cables are all revealing. Lee et al. watch the final maturation of the rat urinary tract by proteomic analysis of the debris found in urine over time. Taking special care not to mix adult and neonatal urine, they examined four samples over 2 weeks after birth and one at maturity, >30 d. Using nano‐ESI‐LC‐MS/MS technology, six proteins were found in all samples, 30 were adult specific. Proteins were further characterized by large format 1‐ and 2‐DE, immunoblots, and immunofluorescent analysis of tissue sections. Days 1, 3, and 7 had 37% of proteins in common whereas days 7, 14 and >30 shared only 7.4% of proteins. Levels of fibronectin and location of E‐cadherin expression shifted during maturation. Lee, R. S. et al., Proteomics 2008, 8, 1097–1112.     

In this issue: Proteomics 5/2008

In this issue of Proteomics you will find the following highlighted articles: When is a stain not a stain? When it's dyeing! [Dumb proteomics joke!] This silly riddle is actually relat­ed to a recurrent question in proteomics: when is the best time to apply detection reagents to proteins for quantitative analysis? (a) pre‐electrophoresis labeling with DIGE/Cy‐type of covalent stains, or (b) post‐electrophoresis staining with silver, Sypro Ruby or Deep Purple? Karp et al. explore the question using a bacterial extract as a typical sample, DIGE Cy labels, and Deep Purple. It gets more complex when they have to deal with the “missingness” of spots: just because a spot doesn’t show up doesn’t mean it is not there, there just may not be enough to detect. Progenesis SameSpots software was used to analyze images for missing spots. In the end, DIGE gave better sensitivity as previously reported, and fewer missing spots. Deep Purple was more competitive when analyzed with SameSpots software. Karp, N. A. et al., Proteomics 2008, 8, 948–960. Your own best enemy? If there weren’t one maverick, black sheep, rebel, outlaw, eccentric, or rotten apple in most families, a lot of novels would never have been written. Mammalian immune systems seem to have the same structure – they mostly target enemies of the body but there always seem to be a few maverick antibodies that are targeted at their own body’s antigens. Servettaz et al. take up proteomic tools to identify the targets of the anti‐self antibodies expressed by apparently healthy individuals. Using umbilical cord endothelial cells as a source of antigens, the authors found 884 spots by ­2‐­DE, and 61 ± 25 of those were recognized by serum IgGs. All 12 sera tested recognized 11 antigens derived from 6 proteins. There were 3 cytoskeletal, 2 glycolytic, and 1 disulfide isomerase protein seen. These were confirmed by immunoblotting of 2‐D gels and identification by in‐gel tryptic digestion and MALDI‐TOF MS. Servettaz, A. et al., Proteomics 2008, 8, 1000–1008. Signature in scraps from kidney growth stages You can tell a lot about the quality of a new building, residential or commercial, by what doesn’t go into it. The scraps of lumber, pieces of masonry, lengths and varieties of cables are all revealing. Lee et al. watch the final maturation of the rat urinary tract by proteomic analysis of the debris found in urine over time. Taking special care not to mix adult and neonatal urine, they examined four samples over 2 weeks after birth and one at maturity, >30 d. Using nano‐ESI‐LC‐MS/MS technology, six proteins were found in all samples, 30 were adult specific. Proteins were further characterized by large format 1‐ and 2‐DE, immunoblots, and immunofluorescent analysis of tissue sections. Days 1, 3, and 7 had 37% of proteins in common whereas days 7, 14 and >30 shared only 7.4% of proteins. Levels of fibronectin and location of E‐cadherin expression shifted during maturation. Lee, R. S. et al., Proteomics 2008, 8, 1097–1112.     

In this issue: Proteomics 7/2008

In this issue of Proteomics you will find the following highlighted articles: Modified amino peptides step out of line, reveal identity In thriller movies and spy stories, you can often tell which character is a bad guy if his “confession” changes under pressure or depends on the inquisitor. Likewise for peptides with modifications. Staes et al. use a similar technique to find α‐amino blocked peptides. After chromatography of a digest over a C₁₈ reverse phase column, fractions were treated with TNBS and re‐chromatographed on the same column, under the same conditions. The peptides that had trypsin‐exposed amino groups became much more hydrophobic in the second round because of the addition of the TNBS. The technique (COFRADIC) was also improved by preceding the C₁₈ column by use of a strong cation exchange for fractionation and using a kit for removal of any pyrrolidone carboxylic acid termini from peptides. The revised protocol raised the yield of true amino termini from 60% to 95%. Staes, A. et al., Proteomics 2008, 8, 1362–1370. Decrypting Cryptosporidium parvum: Proteome data revealed by triple analysis As hikers in North America and normal people in many parts of the world know, Cryptosporidium parvum is a protozoan parasite that causes an unpleasant intestinal infection in humans. It also infects livestock species, which leads to widespread waterborne transmission unless effective water treatment is employed. When the oocytes enter the gastrointestinal tract, they are stimulated to undergo excystation, releasing four sporozoites that enter the epithelial cells. There they undergo asexual reproduction and begin a complex series of steps before reproduction is complete and oocytes are released. Although the genome has been completely sequenced, many of the proteins predicted did not have recognizable functions. Sanderson et al. used a tissue culture system of excystation to collect enough sporozoites for proteomic analysis by MuDPIT and LC‐MS/MS after (a) 2‐DE and (b) 1‐DE. Over 1200 unique proteins were identified, representing >30% of the predicted organism proteome, >200 of which had transmembrane domains. Sanderson, S. J. et al., Proteomics 2008, 8, 1398–1414. Oxidized proteins in serum: Inside job or outside contractor? Reactive oxygen species (ROS) seem to be involved in a variety of diseases, including Alzheimer's, Parkinson's, cancer and heart disease. Searches for biomarkers for these diseases have most commonly been done in blood plasma, which contains proteins from essentially every cell type and tissue in the organism. Mirzaei et al. explore questions of cause and effect in rat plasma by trapping ROS‐caused carbonylation points with biotin hydrazide, followed by avidin affinity chromatography and proteomic analysis (LC‐MS/MS). Of 146 proteins identified in four rats, 44 had at least one carbonylation site and 38 had two or more sites. Over 30% of the proteins were membrane proteins, suggesting a major source of ROS was external, a hypothesis supported by the observation that mitochondrial proteins are not affected, despite their proximity to endogenous ROS. On the other hand, 13% were nuclear proteins. Another surprise: virtually no (2%) plasma proteins were found. Mirzaei, H. et al., Proteomics 2008, 8, 1516–1527.     

Nicotine affects pancreatic cell proteomics across species

While the genome represents a static moiety, the proteome is more dynamic and can change in response to stimuli. As such, proteomics may reveal the effect of various drugs on cellular mechanisms. Protein alterations upon exogenous perturbations are vital in outlining species‐to‐species differences that cannot otherwise be measured quantitatively. Specifically, nicotine has been shown to be an independent risk factor for a multitude of diseases. In pancreatic research its mechanism of action remains unresolved. The pioneering work of Paulo et al. (Proteomics 2013, 13, 1499–1512) is a major step toward understanding the role of nicotine, a principal toxin in cigarette smoke, in pancreatic disease. Equally important, Paulo et al. examine the effect of nicotine on stellate cells across three species, demonstrating the importance of identifying species‐specific effects in translational research.     

Cover Picture: Proteomics 7/2008

In this issue of Proteomics you will find the following highlighted articles: Modified amino peptides step out of line, reveal identity In thriller movies and spy stories, you can often tell which character is a bad guy if his “confession” changes under pressure or depends on the inquisitor. Likewise for peptides with modifications. Staes et al. use a similar technique to find α‐amino blocked peptides. After chromatography of a digest over a C₁₈ reverse phase column, fractions were treated with TNBS and re‐chromatographed on the same column, under the same conditions. The peptides that had trypsin‐exposed amino groups became much more hydrophobic in the second round because of the addition of the TNBS. The technique (COFRADIC) was also improved by preceding the C₁₈ column by use of a strong cation exchange for fractionation and using a kit for removal of any pyrrolidone carboxylic acid termini from peptides. The revised protocol raised the yield of true amino termini from 60% to 95%. Staes, A. et al., Proteomics 2008, 8, 1362–1370. Decrypting Cryptosporidium parvum: Proteome data revealed by triple analysis As hikers in North America and normal people in many parts of the world know, Cryptosporidium parvum is a protozoan parasite that causes an unpleasant intestinal infection in humans. It also infects livestock species, which leads to widespread waterborne transmission unless effective water treatment is employed. When the oocytes enter the gastrointestinal tract, they are stimulated to undergo excystation, releasing four sporozoites that enter the epithelial cells. There they undergo asexual reproduction and begin a complex series of steps before reproduction is complete and oocytes are released. Although the genome has been completely sequenced, many of the proteins predicted did not have recognizable functions. Sanderson et al. used a tissue culture system of excystation to collect enough sporozoites for proteomic analysis by MuDPIT and LC‐MS/MS after (a) 2‐DE and (b) 1‐DE. Over 1200 unique proteins were identified, representing >30% of the predicted organism proteome, >200 of which had transmembrane domains. Sanderson, S. J. et al., Proteomics 2008, 8, 1398–1414. Oxidized proteins in serum: Inside job or outside contractor? Reactive oxygen species (ROS) seem to be involved in a variety of diseases, including Alzheimer's, Parkinson's, cancer and heart disease. Searches for biomarkers for these diseases have most commonly been done in blood plasma, which contains proteins from essentially every cell type and tissue in the organism. Mirzaei et al. explore questions of cause and effect in rat plasma by trapping ROS‐caused carbonylation points with biotin hydrazide, followed by avidin affinity chromatography and proteomic analysis (LC‐MS/MS). Of 146 proteins identified in four rats, 44 had at least one carbonylation site and 38 had two or more sites. Over 30% of the proteins were membrane proteins, suggesting a major source of ROS was external, a hypothesis supported by the observation that mitochondrial proteins are not affected, despite their proximity to endogenous ROS. On the other hand, 13% were nuclear proteins. Another surprise: virtually no (2%) plasma proteins were found. Mirzaei, H. et al., Proteomics 2008, 8, 1516–1527.     

Power of positive thinking in quantitative proteomics

Derivatization of proteins with specific isotope reagents has been widely explored for quantitative proteomics where the relative abundances of proteins present in different complex samples are compared by MS. This represents an interesting arena for innovation, where protein chemistry and MS are associated for the best of both worlds. Among the numerous reagents developed, those that introduce a permanent positive charge, such as (N‐succinimidyloxycarbonylmethyl)‐tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP), increase the ionizability of their targets and thus improve the sensitivity of the approach. TMPP labeling also modifies the hydrophobicity and changes the peptide fragmentation pattern. Because TMPP reacts preferably with the N‐termini of proteins and peptides, its use has been explored for proteogenomics and de novo protein sequencing. In this issue of Proteomics, Shen et al. (Proteomics 2015, 15, 2903–2909) show that accurate quantitation of proteins can be obtained with light/heavy TMPP‐labeling of peptides, which can be easily prepared and desalted in a homemade C8‐SCX‐C8 stagetip, and then monitored by nano‐LC‐MS/MS analysis. Their results demonstrate enhanced sequence coverage compared with other approaches. Combined with an efficient enrichment procedure, the higher sensitivity of this “positive attitude” reagent may facilitate much deeper investigations into the quantitative proteomics of complex samples.     

Peptidome Analysis of Cerebrospinal Fluid by LC-MALDI MS

We report on the analysis of endogenous peptides in cerebrospinal fluid (CSF) by mass spectrometry. A method was developed for preparation of peptide extracts from CSF. Analysis of the extracts by offline LC-MALDI MS resulted in the detection of 3,000-4,000 peptide-like features. Out of these, 730 peptides were identified by MS/MS. The majority of these peptides have not been previously reported in CSF. The identified peptides were found to originate from 104 proteins, of which several have been reported to be involved in different disorders of the central nervous system. These results support the notion that CSF peptidomics may be viable complement to proteomics in the search of biomarkers of CNS disorders.     

Callus induction and plant regeneration from explants of commercial cultivars of leek (Allium ampeloprasum var. porrum L.)

Summary Plant regeneration capacity was studied for 8 cultivars and 4 accessions of leek (A. ampeloprasum var. porrum L.). Compact callus was induced on embryo and leaf explants on three different media. The highest frequency of compact callus formation (up to 90%) was obtained when mature, zygotic embryos were cultured on MS medium, containing 30 g/l sucrose and 1 mg/l 2,4-D. Regeneration occurred through somatic embryogenesis on MS medium, supplemented with 1 mg/l kinetin. Plants could be regenerated from all cultivars and accessions tested. These cultivars and accessions could be classified into three groups with respect to shoot formation frequency. The results suggest a distinct influence of the genotype on the morphogenic response of leek embryo explants in vitro.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium - N₆ medium from Chu et al. (1975) - B₅ medium from Gamborg et al. (1968) - BDS Dunstan and Short medium (1977)     

In this issue: Proteomics 1/2009

In this issue of Proteomics you will find the following highlighted articles: How many tries before you get it right? British Prime Minister Benjamin Disraeli is reputed to have stated that “There are three types of lies: lies, damned lies and statistics.” As those immersed in bioinformatics have recognized, though they may be slippery characters, statistics are the only way some information can be extracted from an experimental structure. One of the recurring problems is the question of how many samples need to be tested to get a reasonable, reliable result. This is particularly important when samples are difficult to get, require arduous preparation, or yield only small amounts. These experiments are generally multidimensional. In this article Cairns et al., examine the number of mass spectrometry samples that are required for a quantitative answer in a biomarker search. They evaluate MALDI‐TOF and SELDI‐TOF data for sources and amounts of variability on a pilot scale (biological and technical particularly) which allows them to calculate the number of samples required for a valid full‐scale screen. Cairns, D. A. et al., Proteomics 2009, 9, 74‐86. Double‐barreled proteomic run on embryonic stem cell membranes Embryonic stem cells (ESC) appear to be as close to the fountain of youth as most of us can reasonably expect to get in this lifetime. How close they come to being a “silver bullet” for cancer and other diseases is yet to be determined. Intoh et al., have taken a major step forward in improving our understanding of ESC control and maintenance. They applied 2‐D DIGE and trypsin digestion + iTRAQ labeling to identify membrane and membrane‐associated proteins in mouse ESCs that had or had not been exposed to leukemia inhibitory factor, a factor which maintains pluripotency in ESCs. Some 338 membrane and membrane‐associated proteins, up‐ or down‐regulated, were identified and assigned to functional groups. Intoh, A. et al., Proteomics 2009, 9, 126‐137. H, M, L You see these three letters on a variety of simple controllers: pump speed, temperature, under‐desk foot warmers, etc. Now you can hope to see them soon on bottles in a cell mass isotope labeling kit. Schwanhäusser et al., describe here a protocol for following levels of protein expression in array volumes and numbers with array simplicity. They pulse label samples with Heavy, Medium, or Light amino acids. Pulse‐labeling has been used for determining protein turnover rates for eons but with a quantitation problem for translation: did the ratio change because the numerator changed or because the denominator changed? The answer comes from labeling the untreated control with the M amino acid, then mixing M+H or M+L samples before fractionating by SDS‐PAGE and high‐resolution LC‐MS/MS. It worked for cell fractions (HeLa) as well as whole cells (yeast). Schwanhäusser, B. et al., Proteomics 2009, 9, 205‐209.     

Cover Picture: Proteomics 1/2009

In this issue of Proteomics you will find the following highlighted articles: How many tries before you get it right? British Prime Minister Benjamin Disraeli is reputed to have stated that “There are three types of lies: lies, damned lies and statistics.” As those immersed in bioinformatics have recognized, though they may be slippery characters, statistics are the only way some information can be extracted from an experimental structure. One of the recurring problems is the question of how many samples need to be tested to get a reasonable, reliable result. This is particularly important when samples are difficult to get, require arduous preparation, or yield only small amounts. These experiments are generally multidimensional. In this article Cairns et al., examine the number of mass spectrometry samples that are required for a quantitative answer in a biomarker search. They evaluate MALDI‐TOF and SELDI‐TOF data for sources and amounts of variability on a pilot scale (biological and technical particularly) which allows them to calculate the number of samples required for a valid full‐scale screen. Cairns, D. A. et al., Proteomics 2009, 9, 74‐86. Double‐barreled proteomic run on embryonic stem cell membranes Embryonic stem cells (ESC) appear to be as close to the fountain of youth as most of us can reasonably expect to get in this lifetime. How close they come to being a “silver bullet” for cancer and other diseases is yet to be determined. Intoh et al., have taken a major step forward in improving our understanding of ESC control and maintenance. They applied 2‐D DIGE and trypsin digestion + iTRAQ labeling to identify membrane and membrane‐associated proteins in mouse ESCs that had or had not been exposed to leukemia inhibitory factor, a factor which maintains pluripotency in ESCs. Some 338 membrane and membrane‐associated proteins, up‐ or down‐regulated, were identified and assigned to functional groups. Intoh, A. et al., Proteomics 2009, 9, 126‐137. H, M, L You see these three letters on a variety of simple controllers: pump speed, temperature, under‐desk foot warmers, etc. Now you can hope to see them soon on bottles in a cell mass isotope labeling kit. Schwanhäusser et al., describe here a protocol for following levels of protein expression in array volumes and numbers with array simplicity. They pulse label samples with Heavy, Medium, or Light amino acids. Pulse‐labeling has been used for determining protein turnover rates for eons but with a quantitation problem for translation: did the ratio change because the numerator changed or because the denominator changed? The answer comes from labeling the untreated control with the M amino acid, then mixing M+H or M+L samples before fractionating by SDS‐PAGE and high‐resolution LC‐MS/MS. It worked for cell fractions (HeLa) as well as whole cells (yeast). Schwanhäusser, B. et al., Proteomics 2009, 9, 205‐209.     

ICPLQuant – A software for non‐isobaric isotopic labeling proteomics

The main goal of many proteomics experiments is an accurate and rapid quantification and identification of regulated proteins in complex biological samples. The bottleneck in quantitative proteomics remains the availability of efficient software to evaluate and quantify the tremendous amount of mass spectral data acquired during a proteomics project. A new software suite, ICPLQuant, has been developed to accurately quantify isotope‐coded protein label (ICPL)‐labeled peptides on the MS level during LC‐MALDI and peptide mass fingerprint experiments. The tool is able to generate a list of differentially regulated peptide precursors for subsequent MS/MS experiments, minimizing time‐consuming acquisition and interpretation of MS/MS data. ICPLQuant is based on two independent units. Unit 1 performs ICPL multiplex detection and quantification and proposes peptides to be identified by MS/MS. Unit 2 combines MASCOT MS/MS protein identification with the quantitative data and produces a protein/peptide list with all the relevant information accessible for further data mining. The accuracy of quantification, selection of peptides for MS/MS‐identification and the automated output of a protein list of regulated proteins are demonstrated by the comparative analysis of four different mixtures of three proteins (Ovalbumin, Horseradish Peroxidase and Rabbit Albumin) spiked into the complex protein background of the DGPF Proteome Marker.     

Immunogold labeling of cryosections from high-pressure frozen cells

Immunogold labeling of cryosections according to Tokuyasu (Tokuyasu KT. A technique for ultracyotomy of cell suspensions and tissues. J Cell Biol 1973;57:551–565), is an important and widely used method for immunoelectron microscopy. These sections are cut from material that is chemically fixed at room temperature (room temparature fixation, RTF). Lately in many morphological studies fast freezing followed by cryosubstitution fixation (CSF) is used instead of RTF. We have explored some new methods for applying immunogold labeling on cryosections from high‐pressure frozen cells (HepG2 cells, primary chondrocytes) and tissues (cartilage and exocrine pancreas). As immunolabeling has to be carried out on thawed and stable sections, we explored two ways to achieve this: (1) The section fixation method, as briefly reported before (Liou W et al. Histochem Cell Biol 1996;106:41–58 and Möbius W et al. J Histochem Cytochem 2002;50:43–55.) in which cryosections from freshly frozen cells were stabilized in mixtures of sucrose and methyl cellulose and varying concentrations of glutaraldehyde, formaldehyde and uranyl acetate (UA). Only occasionally does this method reveal section areas with excellent cell preservation and negatively stained membranes like Tokuyasu sections of RTF material. (Liou et al.) (2) The rehydration method, a novel approach, in which CSF with glutaraldehyde and/or osmium tetroxide (OsO₄) was followed by rehydration and cryosectioning as in the Tokuyasu method. Especially, the addition of UA and low concentrations of water to the CSF medium favored superb membrane contrast. Immunogold labeling was as efficient as with the Tokuyasu method.     

swissPIT: a novel approach for pipelined analysis of mass spectrometry data

The identification and characterization of peptides from tandem mass spectrometry (MS/MS) data represents a critical aspect of proteomics. Today, tandem MS analysis is often performed by only using a single identification program achieving identification rates between 10-50% (Elias and Gygi, 2007). Beside the development of new analysis tools, recent publications describe also the pipelining of different search programs to increase the identification rate (Hartler et al., 2007; Keller et al., 2005). The Swiss Protein Identification Toolbox (swissPIT) follows this approach, but goes a step further by providing the user an expandable multi-tool platform capable of executing workflows to analyze tandem MS-based data. One of the major problems in proteomics is the absent of standardized workflows to analyze the produced data. This includes the pre-processing part as well as the final identification of peptides and proteins. The main idea of swissPIT is not only the usage of different identification tool in parallel, but also the meaningful concatenation of different identification strategies at the same time. The swissPIT is open source software but we also provide a user-friendly web platform, which demonstrates the capabilities of our software and which is available at http://swisspit.cscs.ch upon request for account.