Analysis of intact protein isoforms by mass spectrometry

The diverse proteome of an organism arises from such events as single nucleotide substitutions at the DNA level, different RNA processing, and dynamic enzymatic post-translational modifications. This minireview focuses on the measurement of intact proteins to describe the diversity found in proteomes. The field of biological mass spectrometry has steadily advanced, enabling improvements in the characterization of single proteins to proteins derived from cells or tissues. In this minireview, we discuss the basic technology for "top-down" intact protein analysis. Furthermore, examples of studies involved with the qualitative and quantitative analysis of full-length polypeptides are provided.     

The low resolution structure of ApoA1 in spherical high density lipoprotein revealed by small angle neutron scattering

Spherical high density lipoprotein (sHDL), a key player in reverse cholesterol transport and the most abundant form of HDL, is associated with cardiovascular diseases. Small angle neutron scattering with contrast variation was used to determine the solution structure of protein and lipid components of reconstituted sHDL. Apolipoprotein A1, the major protein of sHDL, forms a hollow structure that cradles a central compact lipid core. Three apoA1 chains are arranged within the low resolution structure of the protein component as one of three possible global architectures: (i) a helical dimer with a hairpin (HdHp), (ii) three hairpins (3Hp), or (iii) an integrated trimer (iT) in which the three apoA1 monomers mutually associate over a portion of the sHDL surface. Cross-linking and mass spectrometry analyses help to discriminate among the three molecular models and are most consistent with the HdHp overall architecture of apoA1 within sHDL.     

Cys-X scanning for expansion of active-site residues and modulation of catalytic functions in a glutathione transferase

We propose Cys-X scanning as a semisynthetic approach to engineer the functional properties of recombinant proteins. As in the case of Ala scanning, key residues in the primary structure are identified, and one of them is replaced by Cys via site-directed mutagenesis. The thiol of the residue introduced is subsequently modified by alternative chemical reagents to yield diverse Cys-X mutants of the protein. This chemical approach is orthogonal to Ala or Cys scanning and allows the expansion of the repertoire of amino acid side chains far beyond those present in natural proteins. In its present application, we have introduced Cys-X residues in human glutathione transferase (GST) M2-2, replacing Met-212 in the substrate-binding site. To achieve selectivity of the modifications, the Cys residues in the wild-type enzyme were replaced by Ala. A suite of simple substitutions resulted in a set of homologous Met derivatives ranging from normethionine to S-heptyl-cysteine. The chemical modifications were validated by HPLC and mass spectrometry. The derivatized mutant enzymes were assayed with alternative GST substrates representing diverse chemical reactions: aromatic substitution, epoxide opening, transnitrosylation, and addition to an ortho-quinone. The Cys substitutions had different effects on the alternative substrates and differentially enhanced or suppressed catalytic activities depending on both the Cys-X substitution and the substrate assayed. As a consequence, the enzyme specificity profile could be changed among the alternative substrates. The procedure lends itself to large-scale production of Cys-X modified protein variants.     

Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease

Polyubiquitin chains on substrates are assembled through any of seven lysine residues or the N terminus of ubiquitin (Ub), generating diverse linkages in the chain structure. PolyUb linkages regulate the fate of modified substrates, but their abundance and function in mammalian cells are not well studied. We present a mass spectrometry-based method to measure polyUb linkages directly from total lysate of mammalian cells. In HEK293 cells, the level of polyUb linkages was found to be 52% (Lys(48)), 38% (Lys(63)), 8% (Lys(29)), 2% (Lys(11)), and 0.5% or less for linear, Lys(6), Lys(27), and Lys(33) linkages. Tissue specificity of these linkages was examined in mice fully labeled by heavy stable isotopes (i.e. SILAC mice). Moreover, we profiled the Ub linkages in brain tissues from patients of Alzheimer disease with or without concurrent Lewy body disease as well as three cellular models of proteolytic stress: proteasome deficiency, lysosome deficiency, and heat shock. The data support that polyUb chains linked through Lys(6), Lys(11), Lys(27), Lys(29), and Lys(48) mediate proteasomal degradation, whereas Lys(63) chains are preferentially involved in the lysosomal pathway. Mixed linkages, including Lys(48), may also contribute to lysosomal targeting, as both Lys(63) and Lys(48) linkages are colocalized in LC3-labeled autophagosomes. Interestingly, heat shock treatment augments Lys(11), Lys(48), and Lys(63) but not Lys(29) linkages, and this unique pattern is similar to that in the profiled neurodegenerative cases. We conclude that different polyUb linkages play distinct roles under the three proteolytic stress conditions, and protein folding capacity in the heat shock responsive pathway might be more affected in Alzheimer disease.     

Three phosphatidylglycerol-phosphate phosphatases in the inner membrane of Escherichia coli

The phospholipids of Escherichia coli consist mainly of phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin. PG makes up ～25% of the cellular phospholipid and is essential for growth in wild-type cells. PG is synthesized on the inner surface of the inner membrane from cytidine diphosphate-diacylglycerol and glycerol 3-phosphate, generating the precursor phosphatidylglycerol-phosphate (PGP). This compound is present at low levels (～0.1% of the total lipid). Dephosphorylation of PGP to PG is catalyzed by several PGP-phosphatases. The pgpA and pgpB genes, which encode structurally distinct PGP-phosphatases, were identified previously. Double deletion mutants lacking pgpA and pgpB are viable and still make PG, suggesting the presence of additional phosphatase(s). We have identified a third PGP-phosphatase gene (previously annotated as yfhB but renamed pgpC) using an expression cloning strategy. A mutant with deletions in all three phosphatase genes is not viable unless covered by a plasmid expressing either pgpA, pgpB, or pgpC. When the triple mutant is covered with the temperature-sensitive plasmid pMAK705 expressing any one of the three pgp genes, the cells grow at 30 but not 42 °C. As growth slows at 42 °C, PGP accumulates to high levels, and the PG content declines. PgpC orthologs are present in many other bacteria.     

Drug metabolite profiling and identification by high-resolution mass spectrometry

Mass spectrometry plays a key role in drug metabolite identification, an integral part of drug discovery and development. The development of high-resolution (HR) MS instrumentation with improved accuracy and stability, along with new data processing techniques, has improved the quality and productivity of metabolite identification processes. In this minireview, HR-MS-based targeted and non-targeted acquisition methods and data mining techniques (e.g. mass defect, product ion, and isotope pattern filters and background subtraction) that facilitate metabolite identification are examined. Methods are presented that enable multiple metabolite identification tasks with a single LC/HR-MS platform and/or analysis. Also, application of HR-MS-based strategies to key metabolite identification activities and future developments in the field are discussed.     

Liquid chromatography-mass spectrometry-based quantitative proteomics

LC-MS-based quantitative proteomics has become increasingly applied to a wide range of biological applications due to growing capabilities for broad proteome coverage and good accuracy and precision in quantification. Herein, we review the current LC-MS-based quantification methods with respect to their advantages and limitations and highlight their potential applications.     

Isolevuglandins and mitochondrial enzymes in the retina: mass spectrometry detection of post-translational modification of sterol-metabolizing CYP27A1

We report the first peptide mapping and sequencing of an in vivo isolevuglandin-modified protein. Mitochondrial cytochrome P450 27A1 (CYP27A1) is a ubiquitous multifunctional sterol C27-hydroxylase that eliminates cholesterol and likely 7-ketocholesterol from the retina and many other tissues. We investigated the post-translational modification of this protein with isolevuglandins, arachidonate oxidation products. Treatment of purified recombinant CYP27A1 with authentic iso[4]levuglandin E(2) (iso[4]LGE(2)) in vitro diminished enzyme activity in a time- and phospholipid-dependent manner. A multiple reaction monitoring protocol was then developed to identify the sites and extent of iso[4]LGE(2) adduction. CYP27A1 exhibited only three Lys residues, Lys(134), Lys(358), and Lys(476), that readily interact with iso[4]LGE(2) in vitro. Such selective modification enabled the generation of an internal standard, (15)N-labeled CYP27A1 modified with iso[4]LGE(2), for the subsequent analysis of a human retinal sample. Two multiple reaction monitoring transitions arising from the peptide AVLK(358)(-C(20)H(26)O(3))ETLR in the retinal sample were observed that co-eluted with the corresponding two (15)N transitions from the supplemented standard. These data demonstrate that modified CYP27A1 is present in the retina. We suggest that such protein modification impairs sterol elimination and likely has other pathological sequelae. We also propose that the post-translational modifications identified in CYP27A1 exemplify a general mechanism whereby oxidative stress and inflammation deleteriously affect protein function, contributing, for example, to cholesterol-rich lesions associated with age-related macular degeneration and cardiovascular disease. The proteomic protocols developed in this study are generally applicable to characterization of lipid-derived oxidative protein modifications occurring in vivo, including proteins bound to membranes.     

Identification of products of inhibition of GES-2 beta-lactamase by tazobactam by x-ray crystallography and spectrometry

The GES-2 β-lactamase is a class A carbapenemase, the emergence of which in clinically important bacterial pathogens is a disconcerting development as the enzyme confers resistance to carbapenem antibiotics. Tazobactam is a clinically used inhibitor of class A β-lactamases, which inhibits the GES-2 enzyme effectively, restoring susceptibility to β-lactam antibiotics. We have investigated the details of the mechanism of inhibition of the GES-2 enzyme by tazobactam. By the use of UV spectrometry, mass spectroscopy, and x-ray crystallography, we have documented and identified the involvement of a total of seven distinct GES-2·tazobactam complexes and one product of the hydrolysis of tazobactam that contribute to the inhibition profile. The x-ray structures for the GES-2 enzyme are for both the native (1.45 Å) and the inhibited complex with tazobactam (1.65 Å). This is the first such structure of a carbapenemase in complex with a clinically important β-lactam inhibitor, shedding light on the structural implications for the inhibition process.