Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been responsible for solving many problems in structural biology. Mass analysis is now used routinely to confirm proper expression and processing of proteins, and to locate and identify post-translational modifications. Innovative advances in instrumentation have led to higher mass resolution and mass accuracy. New sample preparation methods are likewise yielding higher sensitivity plus greater tolerance for buffer components that have in the past suppressed signals at higher concentrations. Advancements in the technique have also led to new or improved applications in many areas, including peptide sequencing and the identification of proteins by database searching with peptide masses. Instruments with lower cost, smaller size, and higher performance are making mass measurements available to an increasing number of laboratories. MALDI-MS is poised to continue to improve in performance and in its usefulness for current and new applications.

GCP5 and GCP6: two new members of the human gamma-tubulin complex.

The gamma-tubulin complex is a large multiprotein complex that is required for microtubule nucleation at the centrosome. Here we report the purification and characterization of the human gamma-tubulin complex and the identification of its subunits. The human gamma-tubulin complex is a ring of ~25 nm, has a subunit structure similar to that reported for gamma-tubulin complexes from other species, and is able to nucleate microtubule polymerization in vitro. Mass spectrometry analysis of the human gamma-tubulin complex components confirmed the presence of four previously identified components (gamma-tubulin and gamma-tubulin complex proteins [GCPs] 2, 3, and 4) and led to the identification of two new components, GCP5 and GCP6. Sequence analysis revealed that the GCPs share five regions of sequence similarity and define a novel protein superfamily that is conserved in metazoans. GCP5 and GCP6, like other components of the gamma-tubulin complex, localize to the centrosome and associate with microtubules, suggesting that the entire gamma-tubulin complex takes part in both of these interactions. Stoichiometry experiments revealed that there is a single copy of GCP5 and multiple copies of gamma-tubulin, GCP2, GCP3, and GCP4 within the gamma-tubulin complex. Thus, the gamma-tubulin complex is conserved in structure and function, suggesting that the mechanism of microtubule nucleation is conserved.

Human seminal relaxin is a product of the same gene as human luteal relaxin.

Unlike that of other species, which have only one gene encoding relaxin, the human genome contains two nonallelic genes for relaxin, designated H1 and H2, which encode markedly different relaxin peptides. Whereas human relaxin gene H2 is selectively expressed in the ovary, no ovarian expression of gene H1 has been detected. Since relaxin is actively produced in the human male, it is possible to postulate divergent gene expression of relaxin in the male and female. We examined this question directly through the structural determination of human seminal relaxin and its comparison with the structure of human luteal relaxin. Partially purified relaxin, prepared from pooled human seminal plasma which had been delipidated by extraction with acid acetone and hexane, subjected to two cycles of HPLC and an additional purification step by ion-exchange chromatography, was further purified by immunoaffinity chromatography, using a monoclonal antibody to the H2 relaxin A chain which cross-reacts with synthetic H1 relaxin, followed by an additional HPLC step performed on a C4 reverse-phase column. The recovered, purified relaxin was then analyzed by N-terminal gas-phase sequencing and fast atom bombardment mass spectroscopy for determination of the amino acid sequence and molecular ions of the A and B chains, respectively. The results demonstrate that the structure of the predominant relaxin in human semen plasma is derived from the product of the H2 gene, consisting of a N-terminal pyroglutamic acid A-24 A chain and a mixture of B-26 and B-27 B chains. With the exception of degradation of the seminal relaxin B chain C-terminus, this structure is identical to the structure of human luteal relaxin. Therefore, both human seminal and luteal relaxin are products of the H2 gene.

Analysis of peptide synthesis products by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry is an easy, rapid method for the verification of proper peptide synthesis and for the identification of most synthetic by-products. A synthesis-purification scheme has been described that uses mass analysis to (1) confirm the presence of the proper product in the crude peptide mixture, (2) guide the purification process, and (3) confirm the mass and purity of the final product. Even though many of these steps could be performed just as well with other ionization techniques, the liquid-flow characteristics of electrospray source are clearly an advantage when LC-MS is required. In addition, the ease with which fragment ions can be generated to provide structural information, even with the least sophisticated instruments, is a further advantage of ESI-MS. Although much of the operation described here was done manually, many of the steps could be automated with little additional effort (e.g., use of an autosampler). Quadrupole and ion trap instruments are widely available at present and provide the chemist with a variety of instruments from which to choose. Electrospray time-of-flight instruments will be commercially have just become available and should also provide similar results. As electrospray instruments continue to evolve, the instruments display greater performance and enhanced user-friendly interfaces, yet are lower in price and smaller in size. These features should lead to even more widespread use for the characterization of synthetic peptides.

Electrospray ionization mass spectrometry of phosphopeptides isolated by on-line immobilized metal-ion affinity chromatography.

Electrospray ionization mass spectrometry (ESI/MS) affords a rapid and sensitive technique for determining peptides produced by the enzymatic digestion of phosphoroteins. When coupled with on-line immobilized metal-ion affinity chromatography (IMAC), the combmation allows separation and mass spectrometric identification of phosphorylated and nonphosphorylated peptides. In this study, the feasibility and general applicability of on-line IMAC/ESI/MS is investigated by using immobilized ferric ions for selective chelation of several phosphotyrosine and phosphoserine peptides. The sensitivity and practicality of the technique for phosphoproteins are demonstrated via the analysis of 30 pmol (∼0.7 µg) of bovine ß-casein purified by sodium dodecylsulfate-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, and digested in situ with trypsin. It is observed that on-line IMAC/ESI/MS suffers less from sample losses than experiments performed off-line, suggesting that the limiting factors in sensitivity for this technique are the purification procedures and sample handling rather than the IMAC and mass spectrometry. Thus, the ability to inject the tryptic digest of an electroblotted protein directly onto the column without buffer exchange and to analyze the eluent directly via on-line coupling of the IMAC column to the mass spectrometer greatly reduces sample losses incurred through sample handling and provides a convenient method for analyzing phosphopeptides at low levels.

Phosphopeptide analysis by on-line immobilized metal-ion affinity chromatography-capillary electrophoresis-electrospray ionization mass spectrometry.

The analysis of large phosphoproteins by mass spectrometry is a particular challenge, in many cases, because of the small proportion of phosphopeptides in the presence of a large number of non-phosphorylated peptides. In addition, phosphopeptides are generally available in dilute solutions. Thus, methods to specifically identify phosphopeptides at low concentrations are important. In this work, on-line Fe(III) immobilized metal-ion affinity chromatography (IMAC)-CE-electrospray ionization MS was developed and applied to sub-pmol analysis of phosphopeptides. Phosphopeptides bind Fe(III) with high selectivity. The IMAC resin is packed directly at the head of the CE column. After the phosphopeptides are bonded to the resin and washed, they are eluted at high pH and separated by CE. This method has several advantages: (1) selective retention and pre-concentration of phosphopeptides on an Fe(III)-IMAC resin; (2) a pre-wash of the sample to remove salts and buffers that are not suited for CE separation or ESI operation; (3) facile fabrication with common tools and chemicals (less than 10 min); (4) adaptation to commercial CE instruments without any modifications. The applications of IMAC-CE-MS are demonstrated by the analysis of phosphopeptide mixtures and a phosphoprotein digest.

Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry.

On-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization-mass spectrometry (IMAC/CE/ESI-MS) offers selective preconcentration of phosphorylated peptides with identification of the phosphorylated amino acid(s). The preconcentration provides low concentration limits of detection and capillary electrophoresis separates the peptides. Recently, we reported a fast, simple, and sensitive on-line IMAC/CE/ESI-MS/MS method for the determination of phosphopeptides at low-pmole levels. That work is expanded here by use of multiple stage tandem mass spectrometry (MS(n), n = 2,3) to isolate and fragment target ions to provide more reliable assignments of phosphorylated residues. The application of IMAC/CE/ESI-MS(n) is demonstrated by the analysis of tryptic digests of alpha- and beta-casein and in-gel tryptic digests of beta-casein.

On the structure and linkage of the covalent cofactor of methylamine dehydrogenase from the methylotrophic bacterium W3A1.

Short amino acid sequences around the two linkage sites of the cofactor of methylamine dehydrogenase are presented. Mass spectral data indicates that the covalently bound cofactor is the tricyclic pyrroloquinoline quinone (PQQ). However, the 3 carboxyl groups characteristic of this o-quinone are absent. A cysteine thioether, via a methylene bridge, and a serine ether link the cofactor to the small subunit of methylamine dehydrogenase.

Tryptic mapping of recombinant proteins by matrix-assisted laser desorption/ionization mass spectrometry.

Various matrix mixtures have been used for matrix-assisted laser desorption/ionization mass spectrometry to characterize the tryptic maps of recombinant human growth hormone (rhGH) and recombinant human tissue plasminogen activator (rt-PA). Carbohydrate-containing comatrices give improved results over single-component matrices. Of those studied, fucose plus 2,5-dihydroxybenzoic acid (DHB) produced a signal for 24 out of 25, or 96%, of the tryptic peptides of rhGH in a single spectrum. These results were obtained for analyses of as little as 280 fmol of unfractionated material measured in digestion buffer. Analysis of 150 fmol showed a decrease in the relative abundance of higher molecular weight peptides. The incorporation of 5-methoxysalicylic acid (5MSA) as a comatrix in a molar ratio of analyte:fucose:DHB:5MSA = 1:5000:5000:50 gave signals for 45 out of 51 peptides for 4.5 pmol of a tryptic digest of rt-PA, corresponding to 88% of the expected fragments. Unobserved peptides were typically di- and tripeptides. Three glycopeptides were observed with peaks corresponding to the known major glycoforms. The fucose/DHB and 5MSA/DHB comatrices produced significant enhancements in spectral quality over DHB alone, including suppression of matrix peaks, increased ion signal, improved resolution, increased number of useful laser shots per crystal, and minimization of baseline slope. Spectra obtained with fucose/DHB generally surpassed DHB/5MSA in quality, though both matrix mixtures were clearly superior to neat DHB. Fucose/DHB demonstrated an increase in tolerance to ionic contaminants by producing a 10-fold reduction in the abundance of (M + Na)+ions. A trimatrix, DHB/5MSA/fucose, produced the highest quality spectra to date, although only marginally better than the fucose/DHB comatrix.

Matrix-assisted laser desorption/ionization time-of-flight mass analysis of peptides.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is one of the most useful techniques for determining the mass of biomolecules, with exceptional capabilities for mass analysis of peptides. Relative to other ionization techniques, it provides high sensitivity and excellent tolerance of salt and other common buffer components. Routine detection limits for peptides are in the subpicomole range. The ions commonly observed are the protonated molecules (M+H(+)), which makes data analysis relatively easy. This overview discusses instrument configuration and calibration, sample preparation, along with specific approaches for analyzing peptide mixtures, synthetic peptides, and chemical modifications of peptides.

Reversed-phase isolation of peptides.

In reversed-phase HPLC, peptides are separated on a hydrophobic stationary phase and eluted with a gradient of increasing organic solvent concentration. Protocols describing the separation of peptides in 5- to 500-pmol quantities via narrow-bore (2-mm-i.d.) or microbore (1-mm-i.d.) columns, as well as for the separation of peptides in quantities <5 pmol are provided in this unit. Capillary HPLC columns require a gradient flow rate of 3 to 5 omponents present in a small sample prior to automated sequencing is possible via the procedures for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and capillary electrophoresis.

Simplification of high-energy collision spectra of peptides by amino-terminal derivatization.

Four-sector tandem mass spectrometry proves extremely useful for providing sequence information for peptides. The complexity of ion fragmentations, however, makes data interpretation difficult and time consuming. Attachment of a fixed positive charge to the peptide amino terminus forces production of only N-terminal fragment ions to yield simplified, predictable fragmentation. Reaction of a peptide at pH 6 with iodoacetic anhydride selectively modifies the N-terminus by exploiting the pK(a) differences between the alpha-amino group and any lysine side-chain epsilon-amino groups. The iodoacetyl peptide can react with many reagents to form a fixed positive charge. We find reaction with dimethyloctylamine forms a quaternary ammonium derivative with good surface activity properties and concomitant increased sensitivity. The high-energy CAD fragment ion spectra of the N-terminally derivatized peptides show predominantly a(n) and d(n) ions. The abundant d(n) ions permit ready distinction of leucine and isoleucine. Fewer fragment ions make data interpretation simpler and lead to more intense peaks since the ion intensity is spread among fewer peaks. The method is particularly useful for peptides which do not otherwise yield sufficient fragmentation to provide either the complete sequence or the locations of modified amino acids.

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.

Analysis of protein digests by capillary high-performance liquid chromatography and on-line fast atom bombardment mass spectrometry.

An HPLC system incorporating a packed capillary C18 column has been utilized for high sensitivity peptide mapping and preparative collection for protein sequencing. This system combined with a Frit-FAB mass spectrometer interface also provides the ability to obtain molecular ions for peptides of enzymatically digested proteins in the time it takes to obtain an HPLC chromatogram. The low flow rates permit introduction of the entire column effluent into the mass spectrometer. Detection limits of 0.5-5 pmol are routine. Proteolytic digests of recombinant human methionyl growth hormone and protein carboxyl methyltransferase have been used to demonstrate the HPLC and mass spectrometer performance.

A general method for highly selective cross-linking of unprotected polypeptides via pH-controlled modification of N-terminal alpha-amino groups.

A method is described for the highly selective modification of the alpha-amino groups at the N-termini of unprotected peptides to form stable, modified peptide intermediates which can be covalently coupled to other molecules or to a solid support. Acylation with iodoacetic anhydride at pH 6.0 occurs with 90-98% selectivity for the alpha-amino group, depending on the N-terminal residue (as shown with a series of model hexapeptides containing a competing Lys residue). Although Cys residues must be protected (reversibly or irreversibly) before the anhydride reaction, there are no detectable side reactions of the alpha-amino moiety--of the reagent or of modified peptide--with the side chains of His, Met, or Lys. The reaction works well in denaturants, so that inhibitory effects of noncovalent structure can be minimized. In a second step the iodoacetyl-peptide can be reacted with a thiol group on a protein, on a solid chromatography matrix, on a spectroscopic probe, etc. This is illustrated by reaction of a series of N alpha-iodoacetyl-peptides with murine interferon-gamma, which contains a C-terminal Cys residue. Data are presented which suggest that this iodoacetic anhydride scheme is superior in selectivity for alpha-amino groups to conventional chemical approaches to cross-linking such as use of 2-iminothiolane or N-hydroxysuccinimide-activated carboxylic acid esters. The reaction is ideally suited for modifying peptide fragments, as pure species or as mixtures, derived from proteolytic or chemical fragmentation of proteins. Furthermore, polypeptides synthesized biosynthetically, for example via recombinant DNA techniques, can be cross-linked in this way.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma desorption mapping of the tryptic digest of 23-kDa recombinant human growth hormone.

Plasma desorption mass spectrometry has been used to map the tryptic fragments from the 23-kDa recombinant human growth hormone protein. The unfractionated tryptic digest was adsorbed directly onto a nitrocellulose sample foil and mass spectra were obtained in both the positive and the negative ion mode. The adsorbed sample was then washed with deionized water and its mass spectrum was again obtained. The latter spectrum revealed tryptic fragments that were not observed in the spectra of the unwashed sample, which can be attributed (to some extent) to the removal of hydrophilic residues during washing. From this study a protocol, aimed at the complete mapping of tryptic fragments, is outlined.

Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption/ionization mass spectrometry of in situ enzymatic digests.

A number of proteins from a silver-stained two-dimensional (2-D) electrophoresis gel of mouse liver whole-cell lysate were identified by peptide mass mapping and sequence database searching. The excised protein spots were processed by in situ reduction and alkylation, followed by Lys-C digestion. The masses of the resulting peptide mixtures were measured with a matrix-assisted laser desorption/ionization (MALDI) reflection-time-of-flight mass spectrometer. These masses were used successfully to search a protein sequence database. Optimized silver staining and digestion protocols allowed proteins to be identified routinely at the low picomole level. The high mass accuracy and resolution provided by delayed extraction were important for high specificity in the database search. Fragment ion data obtained by MALDI post-source decay (PSD) measurements not only provided confirmation of peptide identification, but could be used to identify the protein from a single peptide without spectral interpretation.

Electrospray ionization mass spectrometry of recombinantly engineered antibody fragments.

Mass spectrometry has been used to confirm the correct protein expression of Fab and F(ab')2 fragments of the humanized anti-p185HER2 antibody huMAb4D5. These data demonstrate that electrospray ionization mass spectrometry can be used routinely to measure the masses of purified proteins as large as 100 kDa, with an accuracy of < or = 0.02%. This level of accuracy is helpful in demonstrating the faithful translation and proper posttranslational modification of these proteins. Accurate and reliable mass assignments by electrospray ionization mass spectrometry are achieved by calibrating with protein mass standards, optimizing resolution, and using methods to improve sample purity. On-line reversed-phase high-performance liquid chromatography/mass spectrometry has been employed to improve sample purity and thereby increase sensitivity. Disulfide reduction to yield the component subunits provides additional confirmation of the correct amino acid sequence with greater absolute mass accuracy. A volatile reducing agent such as tributylphosphine provides a convenient method to generate subunits for mass measurement while requiring little or no further sample purification.