A microfabricated device for rapid protein identification by microelectrospray ion trap mass spectrometry

Nanoelectrospray mass spectrometry, the infusion at low flow rates of unseparated peptide mixtures representing protein proteolytic digests into an electrospray ionization mass spectrometer (MS), has been shown to be a suitable method for the analysis of small amounts of proteins. However, the current technique is time consuming, tedious, and difficult to automate. We used microfabrication technologies to construct a device for the sequential infusion of different peptide samples into an electrospray ionization MS without the need for sample manipulation. In this device, etched sample and buffer reservoirs are connected via etched channels to microelectrospray ion source. Peptide samples, typically unseparated tryptic digests of proteins, are applied to different reservoirs. A flow of liquid originating from a specific reservoir is generated and selectively directed toward the microsprayer and the MS by electroosmotic pumping. The analyte proteins are identified by searching sequence databases with the information contained in the collision-induced spectra of selected peptides. With this system, we have achieved a limit of detection in the low femtomoles per microliter range for peptide standards. We also show that samples deposited in different reservoirs can be sequentially mobilized without cross-contamination and that proteins can be conclusively identified at the low femtomoles per microliter level. The successful coupling online of microfabricated devices to an electrospray ionization MS represents an essential step toward the construction of automated, high-throughput, and high-sensitivity analytical systems.     

Protein identification by capillary zone electrophoresis/microelectrospray ionization-tandem mass spectrometry at the subfemtomole level

A method for the identification of proteins by their amino acid sequence at the low-femtomole to subfemtomole sensitivity level is described. It is based on an integrated system consisting of a capillary zone electrophoresis (CZE) instrument coupled to an electrospray ionization triple- quadrupole tandem mass spectrometer (ESI-MS/MS) via a microspray interface. The method consists of proteolytic fragmentation of a protein, peptide separation by CZE, analysis of separated peptides by ESI-MS/MS, and identification of the protein by correlation of the collision-induced dissociation (CID) patterns of selected peptides with the CID patterns predicted from all the isobaric peptides in a sequence database. Using standard peptides applied to a 20-microns-i.d. capillary, we demonstrate an ESI-MS limit of detection of less than 300 amol and CID spectra suitable for searching sequence databases obtained with 600 amol of sample applied to the capillary. Successful protein identification by the method was demonstrated by applying 50 and 38 fmol of a tryptic digest of the proteins beta-lactoglobulin and bovine serum albumin, respectively, to the system.     

Accurate peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Partial 18O-labeling of peptides has been applied to post-source decay experiments in a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The ions which originate from the carboxyl terminus of a peptide partially retain 18O atoms which have readily been incorporated into the C-terminal carboxyl groups during enzymatic hydrolysis in a buffer containing 40 atom percent H218O. The isotopic resolution of singly charged precursor ions and their product ions obtained for peptides up to ca. 2800 Da has been achieved using the delayed extraction method, which permits the rapid identification and assignment of the 18O-labeled and non-labeled ion species in the PSD spectra. The results obtained from several 18O-labeled peptides, derived from an enzymatic digest, demonstrate the accuracy and reproducibility of the present method, which will be in widespread use for protein identification via database searching or for investigations of totally unknown proteins.     

Peptide-mass fingerprinting and the ideal covering set for protein characterisation

The rules that govern the dynamics of protein characterisation by peptide-mass fingerprinting (PMF) were investigated through multiple interrogations of a nonredundant protein database. This was achieved by analysing the efficiency of identifying each entry in the entire database via perfect in silico digestion with a series of 20 pseudo-endoproteinases cutting at the carboxy terminal of each amino acid residue, and the multiple cutters: trypsin, chymotrypsin and Glu-C. The distribution of peptide fragment masses generated by endoproteinase digestion was examined with a view to designing better approaches to protein characterisation by PMF. On average, and for both common and rare cutters, the combination of approximately two fragments was sufficient to identify most database entries. However, the rare cutters left more entries unidentified in the database. Total coverage of the entire database could not be achieved with one enzymatic cutter alone, nor when all 23 cutters were used together. Peptide fragments of > 5000 Da had little effect on the outcome of PMF to correctly characterise database entries, while those with low mass (near to 350 Da in the case of trypsin) were found to be of most utility. The most frequently occurring fragments were also found in this lower mass region. The maximum size of uncut database entries (those not containing a specific amino acid residue) ranged from 52,908 Da to 258,314 Da, while the failure rate for a single cutter in identifying database entries varied from 10,865 (8.4%) to 23,290 (18.1%). PMF is likely to be a mainstay of any high-throughput protein screening strategy for large-scale proteome analysis. A better understanding of the merits and limitations of this technique will allow researchers to optimise their protein characterisation procedures.     

Direct analysis of protein mixtures by tandem mass spectrometry

Methods to identify proteins contained in mixtures are described. The approach uses microcolumn liquid chromatography and automated tandem mass spectrometry in conjunction with protein and nucleotide database searching algorithms. This approach is applied to the identification of proteins obtained by immunoprecipitation reactions, interaction with a GST protein fusion products and interaction with a macromolecular complex.     

Nanoflow solvent gradient delivery from a microfabricated device for protein identifications by electrospray ionization mass spectrometry

Microfabrication technology offers the opportunity to construct microfluidic modules which are designed to perform specific, dedicated functions. Here we report the construction of a microfabricated device for the generation and delivery by electroosmotic pumping of solvent gradients at nanoliter per minute flow rates. The device consists of three solvent reservoirs and channels which were etched in glass. Solvent gradients and solvent flows were generated by computer controlled differential electroosmotic pumping of aqueous and organic phase, respectively, from the solvent reservoirs. The device was integrated into an analytical system consisting of the solvent gradient delivery module, a reverse phase microcolumn and an electrospray ionization ion trap mass spectrometer (MS). The system was used for the analysis at high sensitivity of peptides and peptide mixtures generated by proteolytic digestion of proteins. We have measured an absolute limit of detection as low as 1 fmol and a concentration limit of detection at the 100 amol/microL level. The system was also successfully used for the identification of proteins separated by 1D and 2D gel electrophoresis. This was achieved by gradient frontal analysis of the peptide mixture generated by proteolysis of the respective proteins, and the automated generation and interpretation of collision-induced dissociation spectra.     

Characterisation of proteins from two-dimensional electrophoresis gels by matrix-assisted laser desorption mass spectrometry and amino acid compositional analysis

Amino acid compositional analysis and peptide mass fingerprinting by matrix assisted laser desorption mass spectrometry have been used to characterise proteins obtained from two-dimensional electrophoresis (2-DE) separations of human cardiac proteins. A group of twelve protein spots was selected for analysis. The identities of eight of the proteins had been determined by conventional protein characterisation methods, two were unknown proteins and two had putative identities from protein database spot comparison. Amino acid analysis and peptide mass fingerprinting gave corresponding identities for seven of the twelve proteins, which also agreed with our initial identifications. Three proteins which had been identified previously were not confirmed in this study and putative identities were obtained for the two unknown proteins. The advantages, problems and use of amino acid analysis and peptide mass fingerprinting for the analysis of proteins from 2-DE are discussed. The data highlight the need to use orthogonal techniques for the unequivocal identification of proteins from 2-DE gels.     

From genome to proteome: protein map of Haemophilus influenzae

High-resolution two-dimensional (2-D) polyacrylamide gel electrophoresis allows the separation of complex biological mixtures (i.e., several hundred proteins from a bacterial cell lysate) in a single experiment. In this report proteins from Haemophilus influenzae were separated by 2-D gels and analyzed by peptide mass fingerprinting and/or amino acid analysis. By comparing the peptide mass profiles and the amino acid composition with the Haemophilus influenzae database, 119 protein spots were identified. The combination of amino acid analysis and peptide mass fingerprinting is a powerful tool for a rapid and economical identification of a large number of proteins resolved by 2-D gels. Studies on gene regulation and changes of protein expression upon drug treatment require quick and serial analysis techniques to efficiently identify potential new drug targets.     

Nanoliter chemistry combined with mass spectrometry for peptide mapping of proteins from single mammalian cell lysates

A nanoliter-chemistry station combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was developed to characterize proteins at the attomole level. Chemical reactions including protein digestion were carried out in nanoliter or subnanoliter volumes, followed by microspot sample deposition of the digest to a MALDI-TOF mass spectrometer. Accurate mass determination of the peptides from the enzyme digest, in conjunction with protein database searching, allowed the identification of the proteins in the protein database. This method is particularly useful for handling small-volume samples such as in single-cell analysis. The high sensitivity and specificity of this method were demonstrated by peptide mapping and identifying hemoglobin variants of sickle cell disease from a single red blood cell. The approach of combining nanoliter chemistry with highly sensitive mass spectrometric analysis should find general use in characterizing proteins from biological systems where only a limited amount of material is available for interrogation.     

Sequence database searches via de novo peptide sequencing by tandem mass spectrometry

A method is described for searching protein sequence databases using tandem mass spectra of tryptic peptides. The approach uses a de novo sequencing algorithm to derive a short list of possible sequence candidates which serve as query sequences in a subsequent homology-based database search routine. The sequencing algorithm employs a graph theory approach similar to previously described sequencing programs. In addition, amino acid composition, peptide sequence tags and incomplete or ambiguous Edman sequence data can be used to aid in the sequence determinations. Although sequencing of peptides from tandem mass spectra is possible, one of the frequently encountered difficulties is that several alternative sequences can be deduced from one spectrum. Most of the alternative sequences, however, are sufficiently similar for a homology-based sequence database search to be possible. Unfortunately, the available protein sequence database search algorithms (e.g. Blast or FASTA) require a single unambiguous sequence as input. Here we describe how the publicly available FASTA computer program was modified in order to search protein databases more effectively in spite of the ambiguities intrinsic in de novo peptide sequencing algorithms.     

Capillary electrophoresis/electrospray ionization high mass accuracy time-of-flight mass spectrometry for protein identification using peptide mapping

Capillary electrophoresis/electrospray ionization (CE/ESI) high mass accuracy time-of-flight mass spectrometry was used for the first time to characterize small proteins using peptide mapping. To identify small proteins, the intact proteins were first analyzed to obtain their average molecular weights with errors less than 1 Da. On-line capillary electrophoresis mass spectrometry of the tryptic digests of these small proteins was then performed to obtain the accurate molecular weights of the peptides with accuracies of approximately 10 ppm. Next, this information was used for the identification of the proteins using a protein database. It was found that high mass accuracy is an effective tool in reducing the list of most-likely proteins generated by the database. In addition, on-line collision-induced dissociation of the completely or partially resolved capillary electrophoresis peaks of the protein digests was used to unambiguously identify the sequences of these peptides. Each CE/ESI-MS analysis used only 5 nL of sample containing approximately 120 fmol of each peptide in protein digests. The results indicate that the combination of capillary electrophoresis and high resolution, high mass accuracy time-of-flight mass spectrometry is a viable option for the identification of small proteins using peptide mapping.     

Rapid identification of comigrating gel-isolated proteins by ion trap-mass spectrometry

In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) followed by an initial database search with the peptide masses failed to identify the proteins. The peptides were separated by reversed-phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional sequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence of multiple matching peptide masses in the MALDI-MS spectra. In addition, a novel sequence, not found in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.     

Identification of the components of simple protein mixtures by high-accuracy peptide mass mapping and database searching

Peptide mass mapping by matrix-assisted laser desorption/ionization (MALDI) followed by database searching with the set of measured peptide masses is now a powerful method for the identification of pure proteins. Protein mixtures--such as frequently occur due to comigration in polyacrylamide gel bands--have hitherto required protein sequencing. Here we demonstrate that such protein bands can also be analyzed by peptide mass mapping alone. Database searching with the complete list of peptide masses determined by delayed-extraction MALDI mass spectrometry with a mass error of less than 30 ppm retrieves the most prominent protein in a mixture. In a second step, the protein identity is further confirmed by matching as many of the measured peptide masses as possible to the retrieved amino acid sequence. Peptide masses remaining after this "second pass search" are searched again to identify the next component in the protein mixture. This iterative process is repeated until all major ion signals are accounted for. Protein mixtures consisting of two or more individual components in a single gel band can be analyzed, further increasing the general applicability of MALDI peptide mapping for protein identification.     

On-line coupling of micellar electrokinetic chromatography to electrospray mass spectrometry

The possibility of selectivity enhancement in capillary electrophoresis-mass spectrometry (CE-MS) by hyphenating micellar electrokinetic chromatography (MEKC) and electrospray mass spectrometry (MS) is described for two quaternary ammonium compounds. Direct coupling of MEKC to MS is hazardous because of the contamination of the ion source due to presence of an excess of micelle forming agent in the MEKC buffer. Therefore, a coupled-capillary setup with the possibilities of voltage switching and buffer renewal has been designed. Such a system allows on-line heartcutting of the zones of interest in the MEKC capillary with subsequent transfer via a second capillary to the mass spectrometer.     

Protein identification by solid phase microextraction-capillary zone electrophoresis-microelectrospray-tandem mass spectrometry

We describe an analytical system for the rapid identification of proteins by correlation of tandem mass spectra with protein sequence databases. The system consists of an integrated solid phase microextraction/capillary zone electrophoresis peptide separation device that is connected through a microelectrospray ion source to a tandem mass spectrometer. The limits of detection are 660 amol of sample at a concentration limit of < 33 amol/microliters for peptide mass measurement, and < 10 fmol of sample, at a concentration limit of < 300 amol/microliters for peptide analysis by collision-induced dissociation. Using this system, we have identified low nanogram amounts of yeast proteins separated by high-resolution two-dimensional gel electrophoresis.     

Characterization of serine and threonine phosphorylation sites in beta-elimination/ethanethiol addition-modified proteins by electrospray tandem mass spectrometry and database searching

A new method for the characterization of serine and threonine phosphorylation sites in proteins has been developed. After modification of a phosphoprotein by beta-elimination/ethanethiol addition and conversion of phosphoserine and phosphothreonine residues to S-ethylcysteinyl or beta-methyl-S-ethylcysteinyl residues, the modified protein was subjected to proteolytic digestion. Resulting digests were analyzed by a combination of microbore liquid chromatography, electrospray ionization tandem (MS/MS) ion trap mass spectrometry and database searching to identify original phosphorylated residues. The computer program utilized (SEQUEST) is capable of identifying peptides and modified residues from uninterpreted MS/MS spectra, and using this method, all of the five known phosphorylation sites in bovine beta-casein were identified. Application of the method to multiply phosphorylated human high molecular weight neurofilament protein (NF-H) resulted in the identification of 21 peptides and their modified residues and hence, the in vivo phosphorylation sites. These included 26 KSP and 1 KTP site, all of which occur in the KSP repeat C-terminal tail domain (residues 502-823). One site at residue 518 was previously uncharacterized. A novel non-KSP serine at residue 421 near the KLLEGEE region in a IPFSLPE motif was characterized as phosphorylated (or glycosylated). The 27 characterized phosphorylation sites occur at S/TP residues in the following motifs: KSPVKEE, KSPAEAK, KSPEKEE, KSPAEVK, KSPEKAK, KSPPEAK, KSPVKAE, and KTPAKEE. On the basis of kinase consensus sequences, all of these motifs, including the previously unreported KTPAKEE motif, can be phosphorylated by proline-directed kinases. Advantages of the new method vis-a-vis our previously reported method [Jaffe, H., Veeranna, Shetty, K. T., and Pant, H. C. (1998) Biochemistry 37, 3931-3940] include (i) production of diastereomers eluting at different retention times increased the chances of peptide identification, (ii) increased hydrophobicity and hence retention time of the modified peptides, (iii) facilitation of positive ion production, and (iv) increased susceptibility to tryptic digestion as a result of conversion of negatively charged phosphorylated residues to neutral S-ethylcysteine or beta-methyl-S-ethylcysteine residues.     

Identification of drug metabolites in biological matrices by intelligent automated liquid chromatography/tandem mass spectrometry

A rapid and systematic strategy for the identification of drug metabolites in biological matrices based on liquid chromatography-tandem mass spectrometry (LC/MS/MS) techniques was utilized for the identification of drug metabolites of the HIV protease inhibitor Indinavir. This strategy integrates intelligent realtime mass spectrometry with HPLC detection and a predictive strategy for detecting metabolites arising from common biotransformations, to rapidly elucidate structures of drug metabolites. Structures of metabolites generated from in vitro incubation mixtures of Indinavir were characterized from a single chromatographic analysis using the automated LC/MS/MS methodology, thus reducing data acquisition time and improving efficiency.     

Determination of hepatotoxic pyrrolizidine alkaloids by on-line high performance liquid chromatography mass spectrometry with an electrospray interface

The present study describes the determination of two different types of hepatotoxic pyrrolizidine alkaloids (PAs) and also distinguishing the hepatotoxic PAs from non-toxic ones by both in-source collision-induced dissociation high performance liquid chromatography mass spectrometry (CID-HPLC/MS) and HPLC/MS/MS (CID in the collision cell), using electrospray ionization. The mass spectra provided molecular ions and characteristic fragment ions, which could be used readily for a rapid identification of different types of PAs. Applications of both in-source CID-HPLC/MS and HPLC/MS/MS analytical methods were successful for the determination of PAs in blood samples obtained from rats dosed with PAs and in the PA-containing plant. The results demonstrated that the developed HPLC/MS methods with two different CID techniques provided a very simple and rapid analysis for an unequivocal diagnosis of PA poisoning and for definitive identification of PAs in plants or herbal medicines.     

Towards an automated approach for protein identification in proteome projects

The development of automated, high throughput technologies for the rapid identification of proteins is essential for large-scale proteome projects. While a degree of automation already exists in some stages of the protein identification process, such as automated acquisition of matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectra, efficient interfaces between different stages are still lacking. We report the development of a highly automated, integrated system for large scale identification of proteins separated by two-dimensional gel electrophoresis (2-DE), based on peptide mass fingerprinting. A prototype robotic system was used to image and excise 288 protein spots from an amido black stained polyvinylidene difluoride (PVDF) blot. Protein samples were enzymatically digested with a commercial automated liquid handling system. MALDI-TOF mass spectrometry was used to acquire mass spectra automatically, and the data analysed with novel automated peptide mass fingerprinting database interrogation software. Using this highly automated system, we were able to identify 95 proteins on the basis of peptide mass fingerprinting, isoelectric point and molecular weight, in a period of less than ten working days. Advantages, problems, and future developments in robotic excision systems, liquid handling, and automated database interrogation software are discussed.     

Differentiation of brewing yeast strains by pyrolysis mass spectrometry and Fourier transform infrared spectroscopy

Two rapid spectroscopic approaches for whole-organism fingerprinting--pyrolysis mass spectrometry (PyMS) and Fourier transform infrared spectroscopy (FT-IR)--were used to analyse 22 production brewery Saccharomyces cerevisiae strains. Multivariate discriminant analysis of the spectral data was then performed to observe relationships between the 22 isolates. Upon visual inspection of the cluster analyses, similar differentiation of the strains was observed for both approaches. Moreover, these phenetic classifications were found to be very similar to those previously obtained using genotypic studies of the same brewing yeasts. Both spectroscopic techniques are rapid (typically 2 min for PyMS and 10 s for FT-IR) and were shown to be capable of the successful discrimination of both ale and lager yeasts. We believe that these whole-organism fingerprinting methods could find application in brewery quality control laboratories.