Proteoform-resolved information, obtained by top− down (TD) "intact protein" proteomics, is expected to contribute substantially to the understanding of molecular pathogenic mechanisms and, in turn, identify novel therapeutic and diagnostic targets. However, the robustness of mass spectrometry (MS) analysis of intact proteins in complex biological samples is hindered by the high dynamic range in protein concentration and mass, protein instability, and buffer complexity. Here, we describe an evolutionary step for intact protein investigations through the online implementation of tandem microflow size-exclusion chromatography with nanoflow reversed-phase liquid chromatography and MS (μSEC 2 -nRPLC-MS). Online serial high-/low-pass SEC filtration overcomes the aforementioned hurdles to intact proteomic analysis through automated sample desalting/cleanup and enrichment of target mass ranges (5−155 kDa) prior to nRPLC-MS. The coupling of μSEC to nRPLC is achieved through a novel injection volume control (IVC) strategy of inserting protein trap columns, pre-and post-μSEC columns, to enable injection of dilute samples in high volumes without loss of sensitivity or resolution. Critical characteristics of the approach are tested via rigorous investigations on samples of varied complexity and chemical background. Application of the platform to cerebrospinal fluid (CSF) prefractionated by OFFGEL isoelectric focusing drastically increases the number of intact mass tags (IMTs) detected within the target mass range (5−30 kDa) in comparison to onedimensional nRPLC-MS with approximately 100× less CSF than previous OFFGEL studies. Furthermore, the modular design of the μSEC 2 -nRPLC-MS platform is robust and promises significant flexibility for large-scale TDMS analysis of diverse samples either directly or in concert with other multidimensional fractionation steps.

Proteoform-resolved information, obtained by top-down (TD) ″intact protein″ proteomics, is expected to contribute substantially to the understanding of molecular pathogenic mechanisms and in turn, identify novel therapeutic and diagnostic targets. However, the robustness of mass spectrometry analysis of intact proteins in complex mixtures is hindered by high dynamic range in protein concentration and mass, protein instability, and the chemical complexity of biological samples. Here, we describe an evolutionary step for intact protein investigations through the online implementation of tandem microflow size exclusion chromatography with nanoflow reversed-phase liquid chromatography and mass spectrometry (μSEC2-nRPLC-MS). Online serial high- and low-pass SEC filtration overcomes the aforementioned hurdles to intact proteomic analysis through automated sample desalting/cleanup and enrichment of target mass ranges prior to nRPLC-MS analysis. The coupling of μSEC to nRPLC is achieved through a novel injection volume control (IVC) strategy of inserting protein trap columns pre- and post-μSEC columns to enable injection of dilute samples in high volumes without loss of sensitivity or resolution. Critical characteristics of the approach are tested via rigorous investigations on samples of varied complexity and chemical background. Application to cerebrospinal fluid (CSF) samples pre-fractionated by OFFGEL isoelectric focusing demonstrates that the platform drastically increases the number of intact mass tags detected within the target mass range (< 30 kDa) in comparison to one-dimensional nRPLC-MS starting from approximately 100x less CSF than previous studies. Furthermore, the modular design of the μSEC2-nRPLC-MS platform is robust and promises significant flexibility for large-scale TDMS analysis of diverse samples either directly or in concert with other multidimensional fractionation steps.

The objective of this cross‐sectional study was to evaluate the impact of diet on homocysteine (Hcy), vitamin B12 and folate status in women living in Queretaro (central Mexico). Average mean values for age, weight, height and BMI were 37.6±10.3 ys, 68.4 ± 14.6 kg, and 151.6±5.2 cm and 29.8±6.0 kg/m2, respectively. Mean concentrations of serum vitamin B12, folate and Hcy were 331.5±209.9 pmol/L, 21.1±8.2 nmol/L and 10.9±3.2 nmol/L. Vitamin B12 deficiency was observed in 11.4% of the participants. In addition 21.2% presented marginal status. Only 5.4% of the women were folate deficient. Prevalence of hyperhomocysteinemia was 32.6%. B12 deficient women had inadequate intakes of vitamin B12 in about 50% (<EAR). Increase in Hcy was related to dietary intake and serum concentrations of vitamin B12. Being vitamin B12 deficient increase 2.8 times the risk of presenting hyperhomocysteinemia. Dietary folate was no related to Hcy, but serum values were. Vitamin B12 deficiency as a result of low dietary intake was the most important factor associated to high Hcy in blood in these Mexican women.

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Background Chicago’s HIV epidemic disproportionally affects people of color. Almost a quarter (23%) of these infections occur in Hispanics. It is important to understand sexual behaviors and HIV risk in Latino MSM and transgender women (TGW) to create targeted culturally sensitive harm reduction interventions. However, participation of minority MSM and TGW in survey-based studies is low. The main objective of the study was to understand the sexual health and of Latino MSM and TGW residing in Chicago, Illinois, United States. We herein report subject’s attitudes towards participating in the study and qualitative observations about perceived barriers to enrollment of this population. Methods This study was a cross sectional analysis of a behavioral/HIV seroprevalence survey administered during 2017-2020 to presumed HIV negative, Latino identifying, MSM and TGW individuals. The survey included questions on sexual risk, HIV knowledge and depression scores. We categorized recurrent themes of the most common reasons participants provided for declining to participate in the study. We generated descriptive statistics. Results A total of 48 community organizations assisted with recruitment. Of 149 participants screened, only 18 (12%) agreed to complete the survey. Among those who declined to complete the survey (n=131), the most common reasons given were: participants were uncomfortable answering some questions (n=59, 45.0%), participant’s did not have the time to initiate or complete the survey (n=41, 31.3%) and survey was deemed long (n=16, 12.2%). We categorized barriers to successful recruitment into the following general themes: 1) Participants were unwilling to discuss their sexual history; 2) Participant’s felt uncomfortable taking a rapid oral HIV test; 3) Participant’s lacked transportation; and 3) Participants didn’t have time to complete the survey or thought it was too long. Conclusion Despite extensive community networking, we found barriers to recruitment of high-risk Latino MSM and TGW into an HIV seroprevalence study. Further research is needed to better understand and address these barriers, and thus, increase representation of this key population in prevention studies. Disclosures All Authors: No reported disclosures