Label‐free profiling of skeletal muscle using high‐definition mass spectrometry

We report automated and time‐efficient (2 h per sample) profiling of muscle using ultra‐performance LC coupled directly with high‐definition MS (HDMS^E). Soluble proteins extracted from rat gastrocnemius (n = 10) were digested with trypsin and analyzed in duplicate using a 90 min RPLC gradient. Protein identification and label‐free quantitation were performed from HDMS^E spectra analyzed using Progenesis QI for Proteomics software. In total 1514 proteins were identified. Of these, 811 had at least three unique peptides and were subsequently used to assess the dynamic range and precision of LC‐HDMS^E label‐free profiling. Proteins analyzed by LC‐HDMS^E encompass the entire complement of glycolytic, β‐oxidation, and tricarboxylic acid enzymes. In addition, numerous components of the electron transport chain and protein kinases involved in skeletal muscle regulation were detected. The dynamic range of protein abundances spanned four orders of magnitude. The correlation between technical replicates of the ten biological samples was R² = 0.9961 ± 0.0036 (95% CI = 0.9940 – 0.9992) and the technical CV averaged 7.3 ± 6.7% (95% CI = 6.87 – 7.79%). This represents the most sophisticated label‐free profiling of skeletal muscle to date.     

Synthesis and mass spectrometry analysis of quaternary cryptando‐peptidic conjugates

The bicyclic amines in the form of cryptands, the crown ether analogs, were used in the synthesis of cryptando‐peptidic conjugates with simultaneous formation of quaternary ammonium nitrogen moiety. A series of model cryptando‐peptidic conjugates at the peptide N‐terminus was efficiently prepared by the standard Fmoc solid phase synthesis. Tandem mass spectrometric analysis of the obtained conjugates has shown the specific fragmentation pattern during MS/MS experiment. The obtained cryptandic quaternary ammonium group undergoes the Hofmann elimination during collision‐induced dissociation fragmentation followed by the ethoxyl group elimination. The presented quaternization of cryptands by iodoacetylated peptides is relatively easy and compatible with standard solid‐phase peptide synthesis. Additionally, the applicability of such peptide derivatives and their isotopologues selectively deuterated at the α‐carbon in the quantitative LC‐MS analysis was analyzed. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

Immuno‐MALDI‐TOF MS: New perspectives for clinical applications of mass spectrometry

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI‐TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI‐TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI‐TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.     

Morpheus Spectral Counter: A computational tool for label‐free quantitative mass spectrometry using the Morpheus search engine

Label‐free quantitative MS based on the Normalized Spectral Abundance Factor (NSAF) has emerged as a straightforward and robust method to determine the relative abundance of individual proteins within complex mixtures. Here, we present Morpheus Spectral Counter (MSpC) as the first computational tool that directly calculates NSAF values from output obtained from Morpheus, a fast, open‐source, peptide‐MS/MS matching engine compatible with high‐resolution accurate‐mass instruments. NSAF has distinct advantages over other MS‐based quantification methods, including a greater dynamic range as compared to isobaric tags, no requirement to align and re‐extract MS1 peaks, and increased speed. MSpC features an easy‐to‐use graphic user interface that additionally calculates both distributed and unique NSAF values to permit analyses of both protein families and isoforms/proteoforms. MSpC determinations of protein concentration were linear over several orders of magnitude based on the analysis of several high‐mass accuracy datasets either obtained from PRIDE or generated with total cell extracts spiked with purified Arabidopsis 20S proteasomes. The MSpC software was developed in C# and is open sourced under a permissive license with the code made available at http://dcgemperline.github.io/Morpheus_SpC/ .     

Single‐step protease cleavage elution for identification of protein–protein interactions from GST pull‐down and mass spectrometry

The study of protein–protein interactions is a major theme in biological disciplines. Pull‐down or affinity‐precipitation assays using GST fusion proteins have become one of the most common and valuable approaches to identify novel binding partners for proteins of interest (bait). Non‐specific binding of prey proteins to the beads or to GST itself, however, inevitably complicates and impedes subsequent analysis of pull‐down results. A variety of measures, each with inherent advantages and limitations, can minimise the extent of the background. This technical brief details and tests a modification of established GST pull‐down protocols. By specifically eluting only the bait (minus the GST tag) and the associated non‐specific binding proteins with a simple, single‐step protease cleavage, a cleaner platform for downstream protein identification with MS is established. We present a proof of concept for this method, as evidenced by a GST pull‐down/MS case study of the small guanosine triphosphatase (GTPase) Rab31 in which: (i) sensitivity was enhanced, (ii) a reduced level of background was observed, (iii) distinguishability of non‐specific contaminant proteins from genuine binders was improved and (iv) a putative new protein–protein interaction was discovered. Our protease cleavage step is readily applicable to all further affinity tag pull‐downs.     

Profile of native N‐linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time‐of‐flight mass spectrometry

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N‐linked oligosaccharides released from human serum without derivatization has been developed using on‐line nanoLC and high resolution TOF MS. The N‐linked oligosaccharides were analyzed with MALDI FT‐ICR MS and microchip LC MS (HPLC–Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43×0.075 mm² i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140×0.075 mm² i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N‐linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for ∼96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining ∼4%.     

Intact protein profiling in breast cancer biomarker discovery: Protein identification issue and the solutions based on 3D protein separation,bottom‐up and top‐down mass spectrometry

Proteomics profiling of intact proteins based on MALDI‐TOF MS and derived platforms has been used in cancer biomarker discovery studies. This approach suffers from a number of limitations such as low resolution, low sensitivity, and that no knowledge is available on the identity of the respective proteins in the discovery mode. Nevertheless, it remains the most high‐throughput, untargeted mode of clinical proteomics studies to date. Here we compare key protein separation and MS techniques available for protein biomarker identification in this type of studies and define reasons of uncertainty in protein peak identity. As a result of critical data analysis, we consider 3D protein separation and identification workflows as optimal procedures. Subsequently, we present a new protocol based on 3D LC‐MS/MS with top‐down at high resolution that enabled the identification of HNRNP A2/B1 intact peptide as correlating with the estrogen receptor expression in breast cancer tissues. Additional development of this general concept toward next generation, top‐down based protein profiling at high resolution is discussed.     

Rapid characterization of N‐linked glycans from secreted and gel‐purified monoclonal antibodies using MALDI‐ToF mass spectrometry

Recombinant monoclonal antibodies (MAbs) are increasingly being used for therapeutic use and correct glycosylation of these MAbs is essential for their correct function. Glycosylation profiles are host cell‐ and antibody class‐dependent and can change over culture time and environmental conditions. Therefore, rapid monitoring of glycan addition/status is of great importance for process validity. We describe two workflows of generally applicability for glycan profiling of purified and gel‐purified MAbs produced in NS0 and CHO cells, in which small‐scale antibody purification and buffer exchange is combined with PNGase F glycan cleavage and graphite HyperCarb desalting. MALDI‐ToF mass spectrometry is used for sensitive detection of glycan forms, with the ability to confirm glycan structures by selective ion fragmentation. Both workflows are rapid, technically simple and amenable to automation, and use in multi‐well formats. Biotechnol. Bioeng. 2010;107: 902–908. © 2010 Wiley Periodicals, Inc.     

PAS‐cal: A repetitive peptide sequence calibration standard for MALDI mass spectrometry

Mass spectrometers equipped with matrix‐assisted laser desorption/ionization (MALDI‐MS) require frequent multipoint calibration to obtain good mass accuracy over a wide mass range and across large numbers of samples. In this study, we introduce a new synthetic peptide mass calibration standard termed PAS‐cal tailored for MALDI‐MS based bottom‐up proteomics. This standard consists of 30 peptides between 8 and 37 amino acids long and each constructed to contain repetitive sequences of Pro, Ala and Ser as well as one C‐terminal arginine residue. MALDI spectra thus cover a mass range between 750 and 3200 m/z in MS mode and between 100 and 3200 m/z in MS/MS mode. Our results show that multipoint calibration of MS spectra using PAS‐cal peptides compares well to current commercial reagents for protein identification by PMF. Calibration of tandem mass spectra from LC‐MALDI experiments using the longest peptide, PAS‐cal37, resulted in smaller fragment ion mass errors, more matching fragment ions and more protein and peptide identifications compared to commercial standards, making the PAS‐cal standard generically useful for bottom‐up proteomics.     

Validation of salivary cortisol and testosterone assays in chimpanzees by liquid chromatography‐tandem mass spectrometry

Owing to its high temporal sensitivity, saliva has distinct advantages for measuring steroids, compared with other noninvasive samples such as urine and feces. Here, we report the validity of assaying salivary cortisol (C) and testosterone (T) using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) in captive male chimpanzees, Pan troglodytes. For both the C and T concentrations, we found positive relationships between saliva and plasma. The concentrations of C and T in saliva showed clear patterns of diurnal fluctuation, whereas those in urine and feces did not. These results suggest that the salivary steroid concentrations can be regarded as good indicators of circulating steroid levels. We also developed and validated an efficient method for collecting saliva samples from cotton rope. Although rope includes inherent steroid‐like compounds and may affect the accuracy of steroid measurements, our rope‐washing procedures effectively removed intrinsic steroidal materials. There was a significant association between the C and T concentrations measured from saliva collected from rope licked by the chimpanzees and those measured from saliva collected directly from the mouth. Salivary T values estimated by LC/MS‐MS were similar to those measured by radioimmunoassay. The results indicate the usefulness of saliva as a noninvasive steroid measure and that steroids in the saliva of chimpanzees can be accurately measured by LC‐MS/MS. Am. J. Primatol. 71:696–706, 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of VITEK matrix‐assisted laser desorption ionization–time of flight mass spectrometry for identification of anaerobes

Rapid and adequate identification of anaerobic bacterial species still presents a challenge for most diagnostic laboratories, hindering the selection of appropriate therapy. In this study, the identification capacity of 16S rRNA sequence analysis, VITEK 2 (BioMérieux, Lyon, France) compact analysis and VITEK MS‐mediated identification for anaerobic bacterial species was compared. Eighty‐five anaerobic bacterial isolates from 11 provinces in China belonging to 14 genera were identified by these three methods. Differences in identification between these three methods were compared. Consistent identification results were obtained for 54 (54/85, 63.5%) isolates by all three methods, the most discordant results being concentrated in Clostridium XI (n = 8) and Bacteroides fragilis (n = 9) clusters. Using the VITEK MS system, 74 (74/90, 82.2%) isolates were identified as single species consistent with 16S rRNA sequence analysis, which was significantly better than the results obtained with VITEK 2 Compact (P < 0.01). Misidentifications by the Vitek 2 Compact and Vitek MS systems were mainly observed in the Clostridium XI (n = 8)and B. fragilis clusters (n = 9). VITEK MS identified anaerobic bacteria even after they had been exposed to oxygen for a week. Identification by the Vitek MS system was more consistent with 16S rRNA sequence analysis than identification by Vitek 2 Compact. Continuous expansion of the VITEK MS database with rare described anaerobic species is warranted to improve both the efficiency and accuracy of VITEK MS identification in routine diagnostic microbiology.     

Rapid identification and characterization of Vibrio species using whole‐cell MALDI‐TOF mass spectrometry

Aims: Vibrio identification by means of traditional microbiological methods is time consuming because of the many biochemical tests that have to be performed to distinguish closely related species. This work aimed at evaluating the use of MALDI‐TOF mass spectrometry for the rapid identification of Vibrio (V.) spp. as an advantageous application to rapidly discriminate the most important Vibrio spp. and distinguish Vibrio spp. from closely related bacterial species like Photobacterium damselae and Grimontia hollisae and other aquatic bacteria like Aeromonas spp. Methods and Results: Starting from sub‐colony amounts of pure cultures grown on agar plates, a very simple sample preparation procedure was established and combined with a rapid and automated measurement protocol that allowed species identification within minutes. Closely related species like Vibrio alginolyticus and Vibrio parahaemolyticus or Vibrio cholerae and Vibrio mimicus could thus be differentiated by defining signatures of species‐identifying biomarker ions (SIBIs). As a reference method for species designation and for determination of relationships between strains with molecular markers, partial rpoB gene sequencing was applied. Conclusions: The MALDI‐TOF MS‐based method as well as the rpoB sequence‐based approach for Vibrio identification described in this study produced comparable classification results. The construction of phylogenetic trees from MALDI‐TOF MS and rpoB sequences revealed a very good congruence of both methods. Significance and Impact of the Study: Our results suggest that whole‐cell MALDI‐TOF MS‐based proteometric characterization represents a powerful tool for rapid and accurate classification and identification of Vibrio spp. and related species.     

Investigating the dynamics and polyanion binding sites of fibroblast growth factor‐1 using hydrogen‐deuterium exchange mass spectrometry

In this study, we examined the local dynamics of acidic fibroblast growth factor (FGF‐1) as well as the binding sites of various polyanions including poly‐sulfates (heparin and low MW heparin) and poly‐phosphates (phytic acid and ATP) using hydrogen‐deuterium exchange mass spectrometry (HX‐MS). For local dynamics, results are analyzed at the peptide level as well as in terms of buried amides employing crystallographic B‐factors and compared with a residue level heat map generated from HX‐MS results. Results show that strand 4 and 5 and the turn between them to be the most flexible regions as was previously seen by NMR. On the other hand, the C‐terminal strands 8, 9, and 10 appear to be more rigid which is also consistent with crystallographic B‐factors as well as local dynamics studies conducted by NMR. Crystal structures of FGF‐1 in complex with heparin have shown that heparin binds to N‐terminal Asn18 and to C‐terminal Lys105, Tryp107, Lys112, Lys113, Arg119, Pro121, Arg122, Gln127, and Lys128 indicating electrostatic forces as dominant interactions. Heparin binding as determined by HX‐MS is consistent with crystallography data. Previous studies have also shown that other polyanions including low MW heparin, phytic acid and ATP dramatically increase the thermal stability of FGF‐1. Using HX‐MS, we find other poly anions tested bind in a similar manner to heparin, primarily targeting the turns in the lysine rich C‐terminal region of FGF‐1 along with two distinct N‐terminal regions that contains lysines and arginines/histidines. This confirms the interactions between FGF‐1 and polyanions are primary directed by electrostatics.     

Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis

Despite their important role in regulating gene expression, posttranslational histone modifications remain technically challenging to analyze. For identification by bottom‐up MS, propionylation is required prior to and following trypsin digestion. Hereby, more hydrophobic peptides are generated enabling RP HPLC separation. When histone dynamics are studied in a quantitative manner, specificity, and efficiency of this chemical derivatization are crucial. Therefore we examined eight different protocols, including two different propionylation reagents. This revealed amidation (up to 70%) and methylation (up to 9%) of carboxyl groups as a side reaction. Moreover, incomplete (up to 85%) as well as a specific propionylation (up to 63%) can occur, depending on the protocol. These results highlight the possible pitfalls and implications for data analysis when doing bottom‐up MS on histones.     

Identification of peptide‐binding sites within BSA using rapid,laser‐induced covalent cross‐linking combined with high‐performance mass spectrometry

We are developing a rapid, time‐resolved method using laser‐activated cross‐linking to capture protein‐peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding‐yeast mating pheromone (α‐factor) and the decapeptide human gonadotropin‐releasing hormone (GnRH). Cross‐linking of α‐factor, using a biotinylated, photoactivatable p‐benzoyl‐L‐phenylalanine (Bpa)–modified analog, was energy‐dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA‐peptide complex. The cross‐linked complex was trypsinized and then interrogated with nano‐LC–MS/MS to identify the peptide cross‐links. Cross‐linking was greatly facilitated by Bpa in the peptide, but some cross‐linking occurred at higher laser powers and high concentrations of a non‐Bpa–modified α‐factor. This was supported by experiments using GnRH, a peptide with sequence homology to α‐factor, which was likewise found to be cross‐linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α‐factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser‐activation to facilitate cross‐linking of Bpa‐containing molecules to proteins. The rapid cross‐linking procedure and high performance of MS/MS to identify cross‐links provides a method to interrogate protein‐peptide interactions in a living cell in a time‐resolved manner.     

Over 4100 protein identifications from a Xenopus laevis fertilized egg digest using reversed‐phase chromatographic prefractionation followed by capillary zone electrophoresis–electrospray ionization–tandem mass spectrometry analysis

A tryptic digest generated from Xenopus laevis fertilized embryos was fractionated by RPLC. One set of 30 fractions was analyzed by 100‐min CZE‐ESI‐MS/MS separations (50 h total instrument time), and a second set of 15 fractions was analyzed by 3‐h UPLC‐ESI‐MS/MS separations (45 h total instrument time). CZE‐MS/MS produced 70% as many protein IDs (4134 versus 5787) and 60% as many peptide IDs (22 535 versus 36 848) as UPLC‐MS/MS with similar instrument time (50 h versus 45 h) but with 50 times smaller total consumed sample amount (1.5 μg versus 75 μg). Surprisingly, CZE generated peaks that were 25% more intense than UPLC for peptides that were identified by both techniques, despite the 50‐fold lower loading amount; this high sensitivity reflects the efficient ionization produced by the electrokinetically pumped nanospray interface used in CZE. This report is the first comparison of CZE‐MS/MS and UPLC‐MS/MS for large‐scale eukaryotic proteomic analysis. The numbers of protein and peptide identifications produced by CZE‐ESI‐MS/MS approach those produced by UPLC‐MS/MS, but with nearly two orders of magnitude lower sample amounts.     

ProSight Lite: Graphical software to analyze top‐down mass spectrometry data

Many top‐down proteomics experiments focus on identifying and localizing PTMs and other potential sources of “mass shift” on a known protein sequence. A simple application to match ion masses and facilitate the iterative hypothesis testing of PTM presence and location would assist with the data analysis in these experiments. ProSight Lite is a free software tool for matching a single candidate sequence against a set of mass spectrometric observations. Fixed or variable modifications, including both PTMs and a select number of glycosylations, can be applied to the amino acid sequence. The application reports multiple scores and a matching fragment list. Fragmentation maps can be exported for publication in either portable network graphic (PNG) or scalable vector graphic (SVG) format. ProSight Lite can be freely downloaded from http://prosightlite.northwestern.edu , installs and updates from the web, and requires Windows 7 or a higher version.