High-resolution Top-down Mass Spectrometry Research Articles

Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry.

As histones play central roles in most chromosomal functions including regulation of DNA replication, DNA damage repair, and gene transcription, both their basic biology and their roles in disease development have been the subject of intense study. Because multiple post-translational modifications (PTMs) along the entire protein sequence are potential regulators of histones, a top-down approach, where intact proteins are analyzed, is ultimately required for complete characterization of proteoforms. However, significant challenges remain for top-down histone analysis primarily because of deficiencies in separation/resolving power and effective identification algorithms. Here we used state-of-the-art mass spectrometry and a bioinformatics workflow for targeted data analysis and visualization. The workflow uses ProMex for intact mass deconvolution, MSPathFinder as a search engine, and LcMsSpectator as a data visualization tool. When complemented with the open-modification tool TopPIC, this workflow enabled identification of novel histone PTMs including tyrosine bromination on histone H4 and H2A, H3 glutathionylation, and mapping of conventional PTMs along the entire protein for many histone subunits.

Top-Down Proteomics and Direct Surface Sampling of Neonatal Dried Blood Spots: Diagnosis of Unknown Hemoglobin Variants

We have previously shown that liquid microjunction surface sampling of dried blood spots coupled with high resolution top-down mass spectrometry may be used for screening of common hemoglobin variants HbS, HbC, and HbD. In order to test the robustness of the approach, we have applied the approach to unknown hemoglobin variants. Six neonatal dried blood spot samples that had been identified as variants, but which could not be diagnosed by current screening methods, were analyzed by direct surface sampling top-down mass spectrometry. Both collision-induced dissociation and electron transfer dissociation mass spectrometry were employed. Four of the samples were identified as β-chain variants: two were heterozygous Hb D-Iran, one was heterozygous Hb Headington, and one was heterozygous Hb J-Baltimore. The fifth sample was identified as the α-chain variant heterozygous Hb Phnom Penh. Analysis of the sixth sample suggested that it did not in fact contain a variant. Adoption of the approach in the clinic would require speed in both data collection and interpretation. To address that issue, we have compared manual data analysis with freely available data analysis software (ProsightPTM). The results demonstrate the power of top-down proteomics for hemoglobin variant analysis in newborn samples.

Purification and High-Resolution Top-Down Mass Spectrometric Characterization of Human Salivary α-Amylase

Human salivary α-amylase (HSAMY) is a major component of salivary secretions, possessing multiple important biological functions. Here we have established three methods to purify HSAMY in human saliva for comprehensive characterization of HSAMY by high-resolution top-down mass spectrometry (MS). Among the three purification methods, the affinity method based on the enzyme-substrate specific interaction between amylase and glycogen is preferred, providing the highest purity HSAMY with high reproducibility. Subsequently, we employed Fourier transform ion cyclotron resonance MS to analyze the purified HSAMY. The predominant form of α-amylase purified from saliva of various races and genders is nonglycosylated with the same molecular weight of 55,881.2, which is 1885.8 lower than the calculated value based on the DNA-predicted sequence. High-resolution MS revealed the truncation of the first 15 N-terminal amino acids (-1858.96) and the subsequent formation of pyroglutamic acid at the new N-terminus Gln (-17.03). More importantly, five disulfide bonds in HSAMY were identified (-10.08) and effectively localized by tandem MS in conjunction with complete and partial reduction by tris (2-carboxyethyl) phosphine. Overall, this study demonstrates that top-down MS combined with affinity purification and partial reduction is a powerful method for rapid purification and complete characterization of large proteins with complex and overlapping disulfide bond patterns.

Automethylation of CARM1 allows coupling of transcription and mRNA splicing.

Coactivator-associated arginine methyltransferase 1 (CARM1), the histone arginine methyltransferase and coactivator for many transcription factors, is subject to multiple post-translational modifications (PTMs). To unbiasedly investigate novel CARM1 PTMs we employed high-resolution top-down mass spectrometry. Surprisingly, mouse CARM1 expressed in insect and mammalian expression systems was completely dimethylated at a single site in the C-terminal domain (CTD). We demonstrate that dimethylation of CARM1 occurs both in vivo and in vitro and proceeds via an automethylation mechanism. To probe function of automethylation, we mutated arginine 551 to lysine to create an automethylation-deficient CARM1. Although mutation of CARM1's automethylation site did not affect its enzymatic activity, it did impair both CARM1-activated transcription and pre-mRNA splicing. These results strongly imply that automethylation of CARM1 provides a direct link to couple transcription and pre-mRNA splicing in a manner differing from the other steroid receptor coactivators. Furthermore, our study identifies a self-regulatory signaling mechanism from CARM1's catalytic domain to its CTD.

High Resolution Top-Down MS/MS Reveals Single Amino Acid Sequence Polymorphisms in Rat Cardiac Troponin

Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex. Two of the three subunits, cTnI and cTnT, are expressed only in cardiac muscle and are widely used in the clinic as serum biomarkers of cardiac injury. cTnI and cTnT are subject to extensive post-translational modification such as proteolysis and phosphorylation, but linking modification patterns to function remains a major challenge. In order to obtain a global view of the state of post-translational modification of cTn, we are performing high resolution top-down mass spectrometry on cTn subunits isolated from native tissues. Whole cTn complexes affinity purified from a single rat heart were analyzed in a 7 Tesla Thermo LTQ-FT-ICR mass spectrometer equipped with an ESI source. High resolution MS spectra of cTn from healthy adult rats showed molecular ions for intact cTnT and cTnI as well as phosphorylation and acetylation patterns similar to human cTnI (Zabrouskov et al., 2008 Mol Cell Proteomics, in press). ‘Shadow peaks’ of similar intensity to parent peaks were detected exhibiting masses of cTnI + 16 Da and cTnT + 128 Da, suggestive of single amino acid polymorphisms. Tandem mass spectrometry (MS/MS) analysis by ECD and CAD fragmentation of intact and protease-digested cTn subunits localized an Ala/Ser polymorphism at residue 7 of cTnI, and an additional Gln within a 3 residue alternative splice site beginning at residue 192 of cTnT. High resolution top-down MS/MS has revealed intriguing heterogeneity not only in the extent of phosphorylation, but also in amino acid sequences of cTnI and cTnT even within a single rat heart. Supported by NIH, AHA, UW-CVRC & Wisconsin Partnership for a Healthy Future.

Single amino acid sequence polymorphisms in rat cardiac troponin revealed by top-down tandem mass spectrometry.

Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex in cardiac muscle. Two of the three subunits, cTnI and cTnT, are subject to post-translational modifications such as proteolysis and phosphorylation, but linking modification patterns to function remains a major challenge. To obtain a global view of the biochemical state of cTn in native tissue, we performed high resolution top-down mass spectrometry of cTn heterotrimers from healthy adult rat hearts. cTn heterotrimers were affinity purified, desalted and then directly subjected to mass spectrometry using a 7 Tesla Thermo LTQ-FT-ICR instrument equipped with an ESI source. Molecular ions for N-terminally processed and acetylated cTnI and cTnT were readily detected as were other post-translationally modified forms of these proteins. cTnI was phosphorylated with a distribution of un-, mono- and bisphosphorylated forms of 41 +/- 3%, 46 +/- 1%, 13 +/- 3%, respectively. cTnT was predominantly monophosphorylated and partially proteolyzed at the Glu(29)-Pro(30) peptide bond. Also observed in high resolution spectra were 'shadow' peaks of similar intensity to 'parent' peaks exhibiting masses of cTnI+16 Da and cTnT+128 Da, subsequently shown by tandem mass spectrometry (MS/MS) to be single amino acid polymorphisms. Intact and protease-digested cTn subunits were fragmented by electron capture dissociation or collision activated dissociation to localize an Ala/Ser polymorphism at residue 7 of cTnI. Similar analysis of cTnT localized an additional Gln within a three residue alternative splice site beginning at residue 192. Besides being able to provide unique insights into the global state of post-translational modification of cTn subunits, high resolution top-down mass spectrometry readily revealed naturally occurring single amino acid sequence variants including a genetic polymorphism at residue 7 in cTnI, and an alternative splice isoform that affects a putative hinge region around residue 192 of cTnT, all of which co-exist within a single rat heart.

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High-resolution top-down mass spectrometry was used to characterize eleven integral and five peripheral subunits of the 750 kDa Photosystem II (PSII) complex from the eukaryotic red alga, Galdieria sulphuraria. The primary separation used liquid chromatography mass spectrometry with concomitant fraction collection (LC-MS+) yielding around 40 intact mass tags (IMTs) at 100 ppm mass accuracy on a low-resolution electrospray-ionization mass spectrometer, whose retention and mass were used to guide subsequent high-resolution top-down nano-electrospray Fourier-transform ion-cyclotron resonance mass spectrometry experiments (FT-MS). Both collisionally activated and electron capture dissociation (CAD, ECD) were used to confirm the presence of eleven small subunits to mass accuracy within 5 ppm; PsbE, PsbF, PsbH, PsbI, PsbJ, PsbK, PsbL, PsbM, PsbT, PsbX and PsbZ. All subunits showed covalent modifications that fall into three classes including retention of initiating formylmethionine, removal of methionine at the N-terminus with or without acetylation, and removal of a longer N-terminal peptide. Peripheral subunits identified by top-down analysis included oxygen evolving complex (OEC) subunits PsbO, PsbU, PsbV, as well as Psb28 (PsbW) and Psb27 (‘PsbZ-like’). Top-down high-resolution mass spectrometry provides the necessary precision, typically less than 5 ppm, for identification and characterization of polypeptide composition of these important membrane protein complexes.