Absolute Amount Of Protein Research Articles

Comparison of Relative Activity versus Relative Expression Factors (RAF versus REF) in Predicting Glucuronidation Mediated Drug Clearance Using Recombinant UGTs.

Predicting the quantitative fraction of glucuronidation (fgluc) by individual UDP-glucuronosyltransferase enzymes (UGTs) is challenging due to the lack of selective inhibitors and inconsistent activity of recombinant UGT systems (rUGTs). Our study compares the relative expression versus activity factors (REF versus RAF) to predict fgluc based on rUGT data to human liver and intestinal microsomes (HLM and HIM). REF scalars were derived from a previous in-house proteomics study for eleven UGT enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17), whereas RAF was calculated by measuring activities in rUGTs to microsomes of selective UGT probe substrates. Protein-normalized activity factor (pnAF) values weregenerated after correcting activity of individual UGTs to their corresponding protein abundance. The utility of REF and RAF in predicting fgluc was assessed for three UGT substrates-diclofenac, vorinostat, and raltegravir. The REF values ranged from 0.02 to 1.75, RAF based on activity obtained in rUGTs to HLM/HIM were from 0.1 to 274. pnAF values were ~ 5 to 80-fold, except for UGT2B4 and UGT2B15, where pnAF was ~ 180 and > 1000, respectively. The results revealed confounding effect of differential specific activities (per pmol) of rUGTs in fgluc prediction. The data suggest that the activity of UGT enzymes was significantly lower when compared to their activity in microsomes at the same absolute protein amount (pmol). Collectively, results of this study demonstrate poor and variable specific activity of different rUGTs (per pmol protein), as determined by pnAF values, which should be considered in fgluc scaling.

Squaric Ester-Based Nanogels Induce No Distinct Protein Corona but Entrap Plasma Proteins into their Porous Hydrogel Network.

After intravenous administration of nanocarriers, plasma proteins may rapidly adsorb onto their surfaces. This process hampers the prediction of the nanocarriers' pharmacokinetics as it determines their physiological identity in a complex biological environment. Toward clinical translation it is therefore an essential prerequisite to investigate the nanocarriers' interaction with plasma proteins. Here, this work evaluates a highly "PEGylated" squaric ester-based nanogel with inherent prolonged blood circulation properties. After incubation with human blood plasma, the nanogels are isolated by asymmetrical flow-field flow fractionation. Multiangle light scattering measurements confirm the absence of significant size increases as well as aggregation upon plasma incubation. However, proteomic analyses by gel electrophoresis find minor absolute amounts of proteins (3 wt%), whereas label-free liquid chromatography mass spectrometry identify 65 enriched proteins. Interestingly, the relative abundance of these proteins is almost similar to their proportion in pure native plasma. Due to the nanogels' hydrated and porous network morphology, it is concluded that the detected proteins rather result from passive diffusion into the nanogel network than from specific interactions at the plasma particle interface. Consequently, these results do not indicate a classical surface protein corona but rather reflect the highly outer and inner stealth-like behavior of the porous hydrogel network.

Tissue Sampling and Homogenization with NIRL Enables Spatially Resolved Cell Layer Specific Proteomic Analysis of the Murine Intestine.

For investigating the molecular physiology and pathophysiology in organs, the most exact data should be obtained; if not, organ-specific cell lines are analyzed, or the whole organ is homogenized, followed by the analysis of its biomolecules. However, if the morphological organization of the organ can be addressed, then, in the best case, the composition of molecules in single cells of the target organ can be analyzed. Laser capture microdissection (LCM) is a technique which enables the selection of specific cells of a tissue for further analysis of their molecules. However, LCM is a time-consuming two-dimensional technique, and optimal results are only obtained if the tissue is fixed, e.g., by formalin. Especially for proteome analysis, formalin fixation reduced the number of identifiable proteins, and this is an additional drawback. Recently, it was demonstrated that sampling of fresh-frozen (non-fixed) tissue with an infrared-laser is giving higher yields with respect to the absolute protein amount and number of identifiable proteins than conventional mechanical homogenization of tissues. In this study, the applicability of the infrared laser tissue sampling for the proteome analysis of different cell layers of murine intestine was investigated, using LC–MS/MS-based differential quantitative bottom-up proteomics. By laser ablation, eight consecutive layers of colon tissue were obtained and analyzed. However, a clear distinguishability of protein profiles between ascending, descending, and transversal colon was made, and we identified the different intestinal-cell-layer proteins, which are cell-specific, as confirmed by data from the Human Protein Atlas. Thus, for the first time, sampling directly from intact fresh-frozen tissue with three-dimensional resolution is giving access to the different proteomes of different cell layers of colon tissue.

Genes Encoding Teleost Orthologs of Human Haploinsufficient and Monoallelically Expressed Genes Remain in Duplicate More Frequently Than the Whole Genome.

Gene dosage is an important issue both in cell and evolutionary biology. Most genes are present in two copies or alleles in diploid eukariotic cells. The most outstanding exception is monoallelic gene expression (MA) that concerns genes localized on the X chromosome or in regions undergoing parental imprinting in eutherians, and many other genes scattered throughout the genome. In diploids, haploinsufficiency (HI) implies that a single functional copy of a gene in a diploid organism is insufficient to ensure a normal biological function. One of the most important mechanisms ensuring functional innovation during evolution is whole genome duplication (WGD). In addition to the two WGDs that have occurred in vertebrate genomes, the teleost genomes underwent an additional WGD, after their divergence from tetrapods. In the present work, we have studied on 57 teleost species whether the orthologs of human MA or HI genes remain more frequently in duplicates or returned more frequently in singleton than the rest of the genome. Our results show that the teleost orthologs of HI human genes remained more frequently in duplicate than the rest of the genome in all of the teleost species studied. No signal was observed for the orthologs of genes mapping to the human X chromosome or subjected to parental imprinting. Surprisingly, the teleost orthologs of the other human MA genes remained in duplicate more frequently than the rest of the genome for most teleost species. These results suggest that the teleost orthologs of MA and HI human genes also undergo selective pressures either related to absolute protein amounts and/or of dosage balance issues. However, these constraints seem to be different for MA genes in teleost in comparison with human genomes.

Screening for protein-protein interactions with asymmetrical flow field-flow fractionation.

We describe a new method for screening protein-protein interaction of biopharmaceutical molecules at dilute concentrations to predict development issues at high concentration. The method is based on Asymmetrical Flow Field-Flow Fractionation (AF4) measurements using well known effects of protein-protein attraction on the fractionation profile due to elevated protein concentrations occurring close to the membrane. We explore the effect for 4 different monoclonal antibodies and show that the profiles obtained are quite different. Interestingly, we find that the recovery in AF4 correlates with the diffusion interaction parameter, which is a standard method for the analysis of protein-protein attraction. The results are insensitive to the protein concentration and buffer composition of the sample solution and only depend on the absolute amount of protein loaded and on the running buffer. This makes the method highly suitable for developability assessment in a compound discovery workflow.

Amount of Protein Required to Improve Muscle Mass in Older Adults.

Increased protein intake has been suggested as an effective strategy to treat age-related loss of muscle mass and function, but the amount of protein required to improve muscle and function without exercise in older adults remains unclear. Thus, this secondary data analysis aimed to assess what amount of protein from habitual protein intake was positively associated with changes in muscle mass and gait speed in older women and men. Ninety-six community-dwelling older adults consumed 0.8, 1.2, or 1.5 g/kg/day of protein and maintained their usual physical activity for 12 weeks. Increased protein intake of >0.54 g/kg/day was positively associated with changes in appendicular skeletal muscle mass (ASM)/weight (B = 0.591, p = 0.026), ASM/body mass index (B = 0.615, p = 0.023), and ASM:fat ratio (B = 0.509, p = 0.030) in older men. However, change in protein intake was not associated with change in muscle mass in older women. Additionally, change in protein intake was not associated with change in gait speed in older women and men. The present study suggested that an increased absolute protein amount of >0.54 g/kg/day from habitual protein intake was positively associated with change in muscle mass in older men.

Absolute Protein Amounts and Relative Abundance of Volume-regulated Anion Channel (VRAC) LRRC8 Subunits in Cells and Tissues Revealed by Quantitative Immunoblotting.

The volume-regulated anion channel (VRAC) plays an important role in osmotic cell volume regulation. In addition, it is involved in various physiological processes such as insulin secretion, glia-neuron communication and purinergic signaling. VRAC is formed by hetero-hexamers of members of the LRRC8 protein family, which consists of five members, LRRC8A-E. LRRC8A is an essential subunit for physiological functionality of VRAC. Its obligate heteromerization with at least one of its paralogues, LRRC8B-E, determines the biophysical properties of VRAC. Moreover, the subunit composition is of physiological relevance as it largely influences the activation mechanism and especially the substrate selectivity. However, the endogenous tissue-specific subunit composition of VRAC is unknown. We have now developed and applied a quantitative immunoblot study of the five VRAC LRRC8 subunits in various mouse cell lines and tissues, using recombinant protein for signal calibration. We found tissue-specific expression patterns of the subunits, and generally relative low expression of the essential LRRC8A subunit. Immunoprecipitation of LRRC8A also co-precipitates an excess of the other subunits, suggesting that non-LRRC8A subunits present the majority in hetero-hexamers. With this, we can estimate that in the tested cell lines, the number of VRAC channels per cell is in the order of 10,000, which is in agreement with earlier calculations from the comparison of single-channel and whole-cell currents.

Enhanced Universal Quantification of Biomolecules Using Element MS and Generic Standards: Application to Intact Protein and Phosphoprotein Determination.

Tools that provide absolute quantification of biomolecules, particularly of proteins and their post-translational modifications, without needing suitable specific standards, are urgently demanded nowadays. To this end, we have significantly improved the recently introduced strategy based on CH4 addition to the plasma for absolute quantification of biomolecules using HPLC-ICP-MS. Addition of CO2 has been optimized and finally selected as a safer, more efficient quantitative strategy that is able to provide constant (<6% error) signal response factor for the six elements assayed (S, P, As, Se, Br, I) under compromised conditions. In the particular case of absolute protein quantification, accuracy and precision attainable for S-based absolute determination of intact proteins using internal and external S-generic standards were compared. Potential for real sample analysis was demonstrated by the high-sensitivity analysis of toxins present in snake venoms. Finally, multielemental speciation capabilities of the approach have been also demonstrated through P and S simultaneous analysis in phosphoproteomics. Simultaneous accurate determination of both absolute protein amount and corresponding phosphorylation degree for intact β-casein, and even impurity traces of κ and α-s1 isoforms present, has been successfully achieved using a simple mixture of inorganic P and S standards. The lowest detection limits (<1 fmol protein) ever published for S- and P-based intact protein quantification with ICP-MS are reported.

Absolute Protein Quantification Using AQUA-Calibrated 2D-PAGE.

Absolute protein quantification for the analysis of proteome dynamics is more and more required by the scientific community. Therefore a number of methods have recently been reported that aim at determining concentrations of single proteins in complex samples, all of them having their advantages and limitations. However, for all of these methods an accurate and protein unspecific determination of the total protein amount in a given sample is urgently needed. Here a ninhydrin-based assay established to reach this goal is described. Moreover, an optimized protocol for protein digestion is an inevitable prerequisite for all mass spectrometry-based approaches aiming at absolute protein quantification. In this chapter, various aspects are described which have to be considered during validation of a suitable digestion method and a detailed protocol is presented that can be applied to the digestion of soluble proteins originated from microbes.In order to provide an absolute protein quantification workflow applicable for small scale and large scale approaches, a step-by-step guide is provided for the so-called AQUA-strategy (AQUA=absolute quantification), including selection of suited standard peptides, the development of optimized MS methods and the determination of absolute protein concentration using stable isotope dilution and selected reaction monitoring (SID-SRM). Subsequently, a workflow is introduced that combines targeted mass spectrometry and two-dimensional polyacrylamide gel electrophoresis for the large-scale determination of absolute protein amounts.

Absolute Quantitation of In Vitro Expressed Plant Membrane Proteins by Targeted Proteomics (MRM) for the Determination of Kinetic Parameters.

The purification of a functional soluble protein from biological or in vitro expression systems can be problematic and the enrichment of a functional membrane protein for biochemical analyses can be a serious technical challenge. Recently we have been characterizing plant endomembrane nucleotide sugar transporters using a yeast expression system. However, rather than enriching these in vitro expressed proteins to homogeneity, we have been conducting biochemical characterization of these transport proteins in yeast microsomal fractions. While this approach has enabled us to estimate a variety of kinetic parameters, the accurate determination ofthe turnover number of an enzyme-substrate complex (k cat) requires that the catalytic site concentration (amount of protein) in the total reaction volume is known. As a result, we have been employing targeted proteomics (multiple reaction monitoring) with peptide standards and a triple quadrupole mass spectrometer to estimate the absolute amount of protein in a mixed protein microsomal fraction. The following method details the steps required to define the absolute quantitation of an in vitro expressed membrane protein to define complete kinetic parameters.

Proteolytic activities in cortex of apical parts of Vicia faba ssp. minor seedling roots during kinetin-induced programmed cell death.

Programmed cell death (PCD) is a crucial process in plant development. In this paper, proteolytically related aspects of kinetin-induced PCD in cortex cells of Vicia faba ssp. minor seedlings were examined using morphological, fluorometric, spectrophotometric, and fluorescence microscopic analyses. Cell viability estimation after 46μM kinetin treatment of seedling roots showed that the number of dying cortex cells increased with treatment duration, reaching maximum after 72h. Weight of the apical root segments increased with time and was about 2.5-fold greater after 96h, while the protein content remained unchanged, compared to the control. The total and cysteine-dependent proteolytic activities fluctuated during 1-96-h treatment, which was not accompanied by the changes in the protein amount, indicating that the absolute protein amounts decreased during kinetin-induced PCD. N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), and Z-Leu-Leu-Nva-H (MG115), the respective cysteine, serine, and proteasome inhibitors, suppressed kinetin-induced PCD. PMSF significantly decreased serine-dependent proteolytic activities without changing the amount of proteins, unlike NEM and MG115. More pronounced effect of PMSF over NEM indicated that in the root apical segments, the most important proteolytic activity during kinetin-induced PCD was that of serine proteases, while that of cysteine proteases may be important for protein degradation in the last phase of the process. Both NEM and PMSF inhibited apoptotic-like structure formation during kinetin-induced PCD. The level of caspase-3-like activity of β1 proteasome subunit increased after kinetin treatment. Addition of proteasome inhibitor MG-115 reduced the number of dying cells, suggesting that proteasomes might play an important role during kinetin-induced PCD.

A novel method for quantitative measurements of gene expression in single living cells.

Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. The integrated protein decay reporter allows measuring protein degradation rates in the absence of protein synthesis inhibitors, and in combination with absolute protein levels allows determining absolute amounts of proteins synthesized over the cell cycle. Finally, the internal tag can be excised by inducible expression of Cre recombinase, which enables to estimate endogenous mRNA half-lives. Our method thus opens new avenues in quantitative analysis of gene expression in single living cells.

Large-scale multiplex absolute protein quantification of drug-metabolizing enzymes and transporters in human intestine, liver, and kidney microsomes by SWATH-MS: Comparison with MRM/SRM and HR-MRM/PRM.

The purpose of the present study was to examine simultaneously the absolute protein amounts of 152 membrane and membrane-associated proteins, including 30 metabolizing enzymes and 107 transporters, in pooled microsomal fractions of human liver, kidney, and intestine by means of SWATH-MS with stable isotope-labeled internal standard peptides, and to compare the results with those obtained by MRM/SRM and high resolution (HR)-MRM/PRM. The protein expression levels of 27 metabolizing enzymes, 54 transporters, and six other membrane proteins were quantitated by SWATH-MS; other targets were below the lower limits of quantitation. Most of the values determined by SWATH-MS differed by less than 50% from those obtained by MRM/SRM or HR-MRM/PRM. Various metabolizing enzymes were expressed in liver microsomes more abundantly than in other microsomes. Ten, 13, and eight transporters listed as important for drugs by International Transporter Consortium were quantified in liver, kidney, and intestinal microsomes, respectively. Our results indicate that SWATH-MS enables large-scale multiplex absolute protein quantification while retaining similar quantitative capability to MRM/SRM or HR-MRM/PRM. SWATH-MS is expected to be useful methodology in the context of drug development for elucidating the molecular mechanisms of drug absorption, metabolism, and excretion in the human body based on protein profile information.

Quantification of MFGM proteins in buttermilk and butter serum by means of a stain free SDS-PAGE method

In comparison to skim milk and whey, buttermilk and butter serum contain high amounts of functional milk fat globule membrane (MFGM) proteins. Quantification of MFGM proteins is difficult because no analytical standard material is available. By means of conventional SDS-PAGE with subsequent staining, MFGM proteins are analytically separated. However, due to various protein structures (e.g., glycosylation) the affinity to the dye is inconsistent. A direct correlation between measured band intensity and protein concentration and, therefore, absolute protein quantification is not possible. The purpose of this study was to apply a novel stain free SDS-PAGE technique that relies on measuring UV light fluorescence of tryptophan in order to quantify MFGM proteins. Thus, proteins can be quantified without the need for analytical standards, provided that the relative tryptophan content relative to the molecular weight is known. In this study, buttermilk and butter serum obtained from raw cream were examined and the three most abundant membrane proteins were absolutely quantified. Higher amounts of peripheral MFGM proteins were determined in buttermilk (0.9g/L) and butter serum (2.7g/L), in comparison to integral MFGM proteins (0.3–0.5g/L). Conclusions were drawn regarding the absolute protein amount of MFGM material surrounding native milk fat globules.

Predicting the carcass chemical composition and describing its growth in live pigs of different sexes using computed tomographys

The aims of this study were (1) to evaluate the ability of computed tomography (CT) to predict the chemical composition of live pigs and carcasses, (2) to compare the chemical composition of four different sex types at a commercial slaughter weight and (3) to model and evaluate the chemical component growth of these sex types. A total of 92 pigs (24 entire males (EM), 24 surgically castrated males (CM), 20 immunocastrated males (IM) and 24 females (FE)) was used. A total of 48 pigs (12 per sex type) were scanned repeatedly in vivo using CT at 30, 70, 100 and 120 kg and slaughtered at the end of the experiment. The remaining 44 were CT scanned in vivo and slaughtered immediately: 12 pigs (4 EM, 4 CM and 4 FE) at 30 kg and 16 pigs each at 70 kg and 100 kg (4 per sex type). The left carcasses were CT scanned, and the right carcasses were minced and analysed for protein, fat, moisture, ash, Ca and P content. Prediction equations for the chemical composition were developed using Partial Least Square regression. Allometric growth equations for the chemical components were modelled. By using live animal and carcass CT images, accurate prediction equations were obtained for the fat (with a root mean square error of prediction (RMSEPCV) of 1.31 and 1.34, respectively, and R2=0.91 for both cases) and moisture relative content (g/100 g) (RMSEPCV=1.19 and 1.38 and R2=0.94 and 0.93, respectively) and were less accurate for the protein (RMSEPCV=0.65 and 0.67 and R2=0.54 and 0.63, respectively) and mineral content (RMSEPCV from 0.28 to 1.83 and R2 from 0.09 to 0.62). Better equations were developed for the absolute amounts of protein, fat, moisture and ash (kg) (RMSEPCV from 0.26 to 1.14 and R2 from 0.91 to 0.99) as well as Ca and P (g) (RMSEPCV=144 and 71, and R2=0.76 to 0.66, respectively). At 120 kg, CM had a higher fat and lower moisture content than EM. For protein, CM and IM had lower values than FE and EM. The ash content was higher in EM and IM than in FE and CM, while IM had a higher Ca and P content than the others. The castrated animals showed a higher allometric coefficient for fat and a lower one for moisture, with IM having intermediate values. However, for the Ca and P models, IM presented higher coefficients than EM and FE, and CM were intermediate.

Effective translation of current dietary guidance: understanding and communicating the concepts of minimal and optimal levels of dietary protein

Dietitians and health care providers have critical roles in the translation of the dietary guidance to practice. The protein content of diets for adults can be based on the Recommended Dietary Allowance (RDA) of 0.80 g/kg per day. Alternatively, the most recent Dietary Reference Intakes (DRIs) for macronutrients reflect expanded guidance for assessing protein needs and consider the relative relation of absolute amounts of protein, carbohydrate, and fat to total energy intake in the context of chronic disease prevention. The Acceptable Macronutrient Distribution Range (AMDR) reflects the interrelation between the macronutrients and affords dietitians and clinicians additional flexibility in diet planning. Accounting for the caloric value of RDAs for carbohydrate and fat, "flexible calories" emerge as an opportunity to create varied eating plans that provide for protein intakes in excess of the RDA but within the AMDR. Protein Summit 2.0 highlighted the growing body of scientific evidence documenting the benefits of higher protein intakes at amounts approximating twice the RDA, which include promotion of healthy body weight and preservation of lean body mass and functional ability with age. The essential amino acid (EAA) density of a food also emerged as a novel concept analogous to "nutrient density," which can enable the practitioner to calculate the caloric cost associated with a specific protein source to attain the daily requirement of EAAs to accomplish various health outcomes because these indispensable nutrients have a significant role in protein utilization and metabolic regulation. Tailoring recommendations unique to an individual's varying goals and needs remains a challenge. However, flexibility within the application of DRIs to include consideration of the AMDR provides a sound framework to guide practitioners in effective translation of current dietary guidance with a specific regard for the documented benefits of higher protein intakes.

Current approaches and challenges in targeted absolute quantification of membrane proteins

Experimental determination of absolute protein amounts is becoming increasingly important for the establishment and validation of biomarkers, and systems biology approaches aimed at a quantitative description of a biological process. Residing at compartmental or cellular barriers, and acting as prominent drug targets, integral membranes proteins, being completely embedded in the lipid bilayer, possess characteristic physicochemical properties and are often in low abundance. These features challenge the quantification with targeted MS and the ability to accurately determine the amount of membrane proteins with high sensitivity. This review summarizes the current status of targeted membrane protein quantification with emphasis on sample preparation beforehand MS. From the beginning to the end of a usual sample preparation workflow, consisting essentially of reference point selection, cell lysis, digestion, and addition of suitable isotope-labeled standards, general and particular challenges for membrane proteins will be discussed step by step. Based on the presentation of current achievements, possible measures to better address these challenges and future avenues of targeted membrane proteomics are presented.

Absolute quantification of Corynebacterium glutamicum glycolytic and anaplerotic enzymes by QconCAT.

Determination of absolute protein amounts using quantitative concatemers (QconCAT's) has already been successfully demonstrated for various species including human, animal and yeast. Interestingly, application of the QconCAT methodology for the determination of cytoplasmic enzyme concentrations in a prokaryote has not been described so far. This study is concerned with a novel targeted approach for the absolute quantification of 10 key enzymes from the central carbon metabolism of the industrially important organism Corynebacterium glutamicum. We demonstrate a method that enables complete cell lysis of this robust soil bacterium, thus allowing for accurate quantification of cytoplasmic enzymes. By linking measured enzyme amounts with respective biovolume data, intracellular enzyme concentrations were estimated, which are of special importance for any systems biology approach studying C. glutamicum's metabolism at the mechanistic level. To our knowledge this is the first report of applying the QconCAT methodology for determining intracellular enzyme concentrations in a prokaryote.

Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs.

While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.

Statistical Approach to Protein Quantification

A major goal in proteomics is the comprehensive and accurate description of a proteome. This task includes not only the identification of proteins in a sample, but also the accurate quantification of their abundance. Although mass spectrometry typically provides information on peptide identity and abundance in a sample, it does not directly measure the concentration of the corresponding proteins. Specifically, most mass-spectrometry-based approaches (e.g. shotgun proteomics or selected reaction monitoring) allow one to quantify peptides using chromatographic peak intensities or spectral counting information. Ultimately, based on these measurements, one wants to infer the concentrations of the corresponding proteins. Inferring properties of the proteins based on experimental peptide evidence is often a complex problem because of the ambiguity of peptide assignments and different chemical properties of the peptides that affect the observed concentrations. We present SCAMPI, a novel generic and statistically sound framework for computing protein abundance scores based on quantified peptides. In contrast to most previous approaches, our model explicitly includes information from shared peptides to improve protein quantitation, especially in eukaryotes with many homologous sequences. The model accounts for uncertainty in the input data, leading to statistical prediction intervals for the protein scores. Furthermore, peptides with extreme abundances can be reassessed and classified as either regular data points or actual outliers. We used the proposed model with several datasets and compared its performance to that of other, previously used approaches for protein quantification in bottom-up mass spectrometry.